Enteroaggregative Escherichia Coli Strains Research Articles

Pathogenic characteristics of the Vibrio parahaemolyticus which caused a gastroenteritis outbreak event in Huzhou.

The pathogenic characteristics of V. parahaemolyticus isolated from a gastroenteritis outbreak event in Deqing County of Huzhou City in 2022 were analyzed. Pathogen detection was performed on 30 anal swabs (26 patients, 1 chef and 3 waiters). The isolates of V. parahaemolyticus were analyzed by serum typing, pulsed field gel electrophoresis (PFGE) molecular typing, multiplex fluorescent PCR detection of tdh/trh virulence gene and drug sensitivity test. 15 patients were positive for V. parahaemolyticus, 1 patient was positive for V. parahaemolyticus and Enteroaggregative E. coli (EAEC), 1 patient was positive for EAEC, and the chef was positive for EAEC. The serotype test results of the 16 V. parahaemolyticus were 14 O4:KUT and 2 O10:K4. All samples were negative for other tested bacteria. All V. parahaemolyticus strains were positive for tdh genes and negative for trh gene. The 16 isolates were 100% resistant to ampicillin (AMP), and sensitive to the other12 antibiotics. From the results of serotype and PFGE, the V. parahaemolyticus strains with two serotypes are clustered into two branches according to their serotypes. The three EAEC strains were non-homologous. In conclusion, we detected V. parahaemolyticus and EAEC from an outbreak of gastroenteritis. And V. parahaemolyticus with two serotypes may be the cause of this event, according to the traceability results.

Remarkable antibiofilm activity of ciprofloxacin, cefoxitin, and tobramycin, by themselves or in combination, against enteroaggregative Escherichia coli in vitro

Enteroaggregative Escherichia coli (EAEC), a biofilm forming pathogen, causes acute and persistent diarrhea worldwide, requiring antimicrobial therapy in severe or persistent cases. To determine the susceptibility of EAEC biofilm to antimicrobials, as single-agent or combined therapy, biofilm formation was investigated using EAEC clinical strains via peg lid. Of the 78 initially analyzed strains, 35 could form biofilms, 15 (42.9%; 15/35) were resistant to at least 1 tested antimicrobial and 20 (57.1%) were susceptible to all of them in the planktonic form. The biofilms of these susceptible strains were challenged against chosen antimicrobials, and displayed resistance to tetracycline, trimethoprim-sulfamethoxazole, chloramphenicol, ampicillin, cefotaxime, ceftriaxone (85%–100%), tobramycin (25%), cefoxitin (20%), and ciprofloxacin (5%). Moreover, ciprofloxacin combined with ampicillin, and tobramycin eradicated the biofilm of 2 of the 4 tested strains. Ciprofloxacin, cefoxitin, and tobramycin maintained their activity well against EAEC biofilm, suggesting their possible effectiveness to treat diarrhea caused by biofilm-forming EAEC strains.

In Vitro Effect of the Cell-Free Supernatant of the Lactobacillus casei Strain IMAU60214 against the Different Pathogenic Properties of Diarrheagenic Escherichia coli

Enteroaggregative Escherichia coli (EAEC) and enterohemorrhagic E. coli (EHEC) are E. coli pathotypes associated with unmanageable diarrhea in children and adults. An alternative to the treatment of infections caused by these microorganisms is the use of the bacteria of the Lactobacillus genus; however, the beneficial effects on the intestinal mucosa are specific to the strain and species. The interest of this study consisted of analyzing the coaggregation properties of Lactobacillus casei IMAU60214, as well as the effect of cell-free supernatant (CSF) on growth and anti-cytotoxic activity in a cell model of the human intestinal epithelium for an agar diffusion assay (HT-29) and the inhibition of biofilm formation on plates of DEC strains of the EAEC and EHEC pathotypes. The results showed that L. casei IMAU60214 exhibits time-dependent coaggregation (35–40%) against EAEC and EHEC that is similar to the control E. coli ATCC 25922. The CSF showed antimicrobial activity (20–80%) against EAEC and EHEC depending on the concentration. In addition, the formation and dispersion of biofilms of the same strains decrease, and the proteolytic pre-treatment with catalase and/or proteinase K (1 mg/mL) of CSF reduces the antimicrobial effect. When evaluating the effect in HT-29 cells pre-treated with CFS on the toxic activity induced by the EAEC and EHEC strains, a reduction of between 30 and 40% was observed. The results show that L. casei IMAU60214 and its CSF have properties that interfere with some properties associated with the virulence of the EAEC and EHEC strains that cause intestinal infection, which supports their use for the control and prevention of infections caused by these bacteria.

Antimicrobial resistance and plasmid-mediated colistin resistance mechanism of diarrheagenic Escherichia coli recovered from foods in parts of China in 2020

Objective: To investigate the antimicrobial resistance of food-borne diarrheagenic Escherichia coli (DEC) and the prevalence of mcr genes that mediates mobile colistin resistance in parts of China, 2020. Methods: For 91 DEC isolates recovered from food sources collected from Fujian province, Hebei province, Inner Mongolia Autonomous Region and Shanghai city in 2020, Vitek2 Compact biochemical identification and antimicrobial susceptibility testing platform was used for the detection of antimicrobial susceptibility testing (AST) against to 18 kinds of antimicrobial compounds belonging to 9 categories, and multi-polymerase chain reaction (mPCR) was used to detect the mcr-1-mcr-9 genes, then a further AST, whole genome sequencing (WGS) and bioinformatics analysis were platformed for these DEC isolates which were PCR positive for mcr genes. Results: Seventy in 91 isolates showed different antimicrobial resistance levels to the drugs tested with a resistance rate of 76.92%. The isolates showed the highest antimicrobial resistance rates to ampicillin (69.23%, 63/91) and trimethoprim-sulfamethoxazole (59.34%, 54/91), respectively. The multiple drug-resistant rate was 47.25% (43/91). Two mcr-1 gene and ESBL (extended-spectrum beta-lactamase) positive EAEC (enteroaggregative Escherichia coli) strains were detected. One of them was identified as serotype of O11:H6, which showed a resistance profile to 25 tested drugs referring to 10 classes, and 38 drug resistance genes were predicted by genome analysis. The other one was O16:H48 serotype, which was resistant to 21 tested drugs belonging to 7 classes and carried a new variant of mcr-1 gene (mcr-1.35). Conclusion: An overall high-level antimicrobial resistance was found among foodborne DEC isolates recovered from parts of China in 2020, and so was the MDR (multi-drug resistance) condition. MDR strains carrying multiple resistance genes such as mcr-1 gene were detected, and a new variant of mcr-1 gene was also found. It is necessary to continue with a dynamic monitoring on DEC contamination and an ongoing research into antimicrobial resistance mechanisms.

Adhesin related genes as potential markers for the enteroaggregative Escherichia coli category.

Enteroaggregative Escherichia coli (EAEC) is an important cause of diarrhea in children and adults worldwide. This pathotype is phenotypically characterized by the aggregative-adherence (AA) pattern in HEp-2 cells and genetically associated to the presence of the aatA gene. EAEC pathogenesis relies in different virulence factors. At least, three types of adhesins have been specifically associated with EAEC strains: the five variants of the aggregative adherence fimbriae (AAF), the aggregative forming pilus (AFP) and more recently, a fibrilar adhesin named CS22. Our study aimed to evaluate the presence of AAF, AFP and CS22-related genes among 110 EAEC strains collected from feces of children with diarrhea. The presence of aggR (EAEC virulence regulator) and genes related to AAFs (aggA, aafA, agg3A, agg4A, agg5A and agg3/4C), AFP (afpA1 and afpR) and CS22 (cseA) was detected by PCR, and the adherence patterns were evaluated on HeLa cells. aggR-positive strains comprised 83.6% of the collection; among them, 80.4% carried at least one AAF-related gene and presented the AA pattern. aggA was the most frequent AAF-related gene (28.4% of aggR+ strains). cseA was detected among aggR+ (16.3%) and aggR- strains (22.2%); non-adherent strains or strains presenting AA pattern were observed in both groups. afpR and afpA1 were exclusively detected among aggR- strains (77.8%), most of which (71.4%) also presented AA pattern. Our results indicate that AAF- and AFP-related genes may contribute to identify EAEC strains, while the presence of cseA and its importance as an EAEC virulence factor and genotypic marker needs to be further evaluated.

Analysis of the Virulence and Inflammatory Markers Elicited by Enteroaggregative Escherichia coli Isolated from Clinical and Non-Clinical Sources in an Experimental Infection Model, India

Enteroaggregative Escherichia coli (EAEC) is highly heterogeneous in virulence; we wanted to understand the pathogenic potential of EAEC isolated from various clinical and non-clinical sources in an animal model. We infected male BALB/c mice in six mice/groups with 50 EAEC isolates isolated from clinical and non-clinical sources. We studied colonization, weight loss, stool shedding, and inflammatory markers and their relationship with 21 virulence genes and phylogroups, EAEC organ burden, and histopathological changes. We detected significantly more inflammatory changes and fecal lactoferrin and calprotectin levels in mice infected with EAEC isolated from symptomatic cases. In clinical EAEC isolates, the presence of chromosomal genes (aap (46%), aaiC (23.3%), SPATEs (pet (13.3%), sat (20%), sigA, and pic (6.6%)), the adhesive variantsof EAEC (agg4A (53.3%), aggA (53.3%), aafA (36.6%), andagg3A (40%)), and the master regulator gene aggR (66.6%) were associated with higher levels of lactoferrin and calprotectin. Additionally, 70% (9/13) of EAEC isolated from acute diarrheal cases bearing chuA (70%) in our study were assigned to groups B2 (4 isolates) and D (5 isolates). Real-time PCR analysis revealed that colonization by EAEC strains from different clinical and non-clinical sources occurs up to 10–15 days of life. Even from non-diarrheal stools and non-clinical sources, EAEC strainshad the potential to cause prolonged colonization, weight loss, and inflammation in the intestine, though the degree varied. Moreover, a better understanding of EAEC pathogenic pathways is desperately needed in different clinical scenarios.

Antimicrobial Efficacy of Green Synthesized Nanosilver with Entrapped Cinnamaldehyde against Multi-Drug-Resistant Enteroaggregative Escherichia coli in Galleria mellonella.

The global emergence of antimicrobial resistance (AMR) needs no emphasis. In this study, the in vitro stability, safety, and antimicrobial efficacy of nanosilver-entrapped cinnamaldehyde (AgC) against multi-drug-resistant (MDR) strains of enteroaggregative Escherichia coli (EAEC) were investigated. Further, the in vivo antibacterial efficacy of AgC against MDR-EAEC was also assessed in Galleria mellonella larval model. In brief, UV-Vis and Fourier transform infrared (FTIR) spectroscopy confirmed effective entrapment of cinnamaldehyde with nanosilver, and the loading efficiency was estimated to be 29.50 ± 0.56%. The AgC was of crystalline form as determined by the X-ray diffractogram with a mono-dispersed spherical morphology of 9.243 ± 1.83 nm in electron microscopy. AgC exhibited a minimum inhibitory concentration (MIC) of 0.008–0.016 mg/mL and a minimum bactericidal concentration (MBC) of 0.008–0.032 mg/mL against MDR- EAEC strains. Furthermore, AgC was stable (high-end temperatures, proteases, cationic salts, pH, and host sera) and tested safe for sheep erythrocytes as well as secondary cell lines (RAW 264.7 and HEp-2) with no negative effects on the commensal gut lactobacilli. in vitro, time-kill assays revealed that MBC levels of AgC could eliminate MDR-EAEC infection in 120 min. In G. mellonella larvae, AgC (MBC values) increased survival, decreased MDR-EAEC counts (p < 0.001), had an enhanced immunomodulatory effect, and was tested safe to the host. These findings infer that entrapment enhanced the efficacy of cinnamaldehyde and AgNPs, overcoming their limitations when used individually, indicating AgC as a promising alternative antimicrobial candidate. However, further investigation in appropriate animal models is required to declare its application against MDR pathogens.

Novel organisation and regulation of the pic promoter from enteroaggregative and uropathogenic Escherichia coli

ABSTRACT The serine protease autotransporters of the Enterobacteriaceae (SPATEs) are a large family of virulence factors commonly found in enteric bacteria. These secreted virulence factors have diverse functions during bacterial infection, including adhesion, aggregation and cell toxicity. One such SPATE, the Pic mucinase (protein involved in colonisation) cleaves mucin, allowing enteric bacterial cells to utilise mucin as a carbon source and to penetrate the gut mucus lining, thereby increasing mucosal colonisation. The pic gene is widely distributed within the Enterobacteriaceae, being found in human pathogens, such as enteroaggregative Escherichia coli (EAEC), uropathogenic E. coli (UPEC) and Shigella flexneri 2a. As the pic promoter regions from EAEC strain 042 and UPEC strain CFT073 differ, we have investigated the regulation of each promoter. Here, using in vivo and in vitro techniques, we show that both promoters are activated by the global transcription factor, CRP (cyclic AMP receptor protein), but the architectures of the EAEC and the UPEC pic promoter differ. Expression from both pic promoters is repressed by the nucleoid-associated factor, Fis, and maximal promoter activity occurs when cells are grown in minimal medium. As CRP activates transcription in conditions of nutrient depletion, whilst Fis levels are maximal in nutrient-rich environments, the regulation of the EAEC and UPEC pic promoters is consistent with Pic’s nutritional role in scavenging mucin as a suitable carbon source during colonisation and infection.

Comparative efficiency of carbohydrates on the biofilm‐forming ability of enteroaggregative Escherichia coli

AbstractThe biofilm‐forming ability of microbes is often correlated with drug resistance; however, this association among enteroaggregative Escherichia coli (EAEC) strains remains naïve. Moreover, the investigations exploring uptake and assimilation of sugars either qualitatively or quantitatively by the biofilm‐forming EAEC strains have not yet been documented. Hence, this study investigated the role of various categories of sugars (monosaccharides, disaccharides, and polysaccharide) on the biofilm‐forming ability of three each of multi‐drug‐resistant (MDR) as well as non‐MDR typical EAEC strains. The EAEC strains exhibited black colonies on congo red agar (CRA) with monosaccharides and disaccharides, whereas all the strains exhibited red colonies on CRA with the polysaccharide. Although significant (p &lt; 0.001) biofilm‐forming ability was observed by all the EAEC strains in sugar‐dependent microtiter plate assay, color change from pink to orange and yellow was noticed in presence of monosaccharides and disaccharide, while absent in polysaccharides. Besides, significantly higher bacterial counts were observed in presence of monosaccharides and disaccharides (p &lt; 0.01); however, such significant bacterial counts were not noticed with the polysaccharide. Further, the real‐time PCR data exhibited comparatively higher relative expression of biofilm‐associated fis gene among EAEC strains grown in presence of monosaccharides, followed by disaccharide and polysaccharide, respectively that concurred with the flow cytometry data. No significant (p &gt; 0.05) difference was observed in the biofilm‐forming ability between MDR‐ and non‐MDR EAEC strains. This study illustrated the role of carbohydrates on the biofilm‐forming ability of EAEC and warranted utmost attention concerning the usage of carbohydrates in food processing industries, for its incorporation as functional food additives.

The aggregate-forming pili (AFP) mediates the aggregative adherence of a hybrid-pathogenic Escherichia coli (UPEC/EAEC) isolated from a urinary tract infection

ABSTRACT Enteroaggregative Escherichia coli (EAEC) comprises an important diarrheagenic pathotype, while uropathogenic E. coli (UPEC) is the most important agent of urinary tract infection (UTI). Recently, EAEC virulence factors have been detected in E. coli strains causing UTI, showing the importance of these hybrid-pathogenic strains. Previously, we detected an E. coli strain isolated from UTI (UPEC-46) presenting characteristics of EAEC, e.g., the aggregative adherence (AA) pattern and EAEC-associated genes (aatA, aap, and pet). In this current study, we analyzed the whole genomic sequence of UPEC-46 and characterized some phenotypic traits. The AA phenotype was observed in cell lineages of urinary and intestinal origin. The production of curli, cellulose, bacteriocins, and Pet toxin was detected. Additionally, UPEC-46 was not capable of forming biofilm using different culture media and human urine. The genome sequence analysis showed that this strain belongs to serotype O166:H12, ST10, and phylogroup A, harbors the tet, aadA, and dfrA/sul resistance genes, and is phylogenetically more related to EAEC strains isolated from human feces. UPEC-46 harbors three plasmids. Plasmid p46-1 (~135 kb) carries some EAEC marker genes and those encoding the aggregate-forming pili (AFP) and its regulator (afpR). A mutation in afpA (encoding the AFP major pilin) led to the loss of pilin production and assembly, and notably, a strongly reduced adhesion to epithelial cells. In summary, the genetic background and phenotypic traits analyzed suggest that UPEC-46 is a hybrid strain (UPEC/EAEC) and highlights the importance of AFP adhesin in the adherence to colorectal and bladder cell lines.

Modulation of AggR levels reveals features of virulence regulation in enteroaggregative E. coli

Enteroaggregative Escherichia coli (EAEC) strains are one of the diarrheagenic pathotypes. EAEC strains harbor a virulence plasmid (pAA2) that encodes, among other virulence determinants, the aggR gene. The expression of the AggR protein leads to the expression of several virulence determinants in both plasmids and chromosomes. In this work, we describe a novel mechanism that influences AggR expression. Because of the absence of a Rho-independent terminator in the 3′UTR, aggR transcripts extend far beyond the aggR ORF. These transcripts are prone to PNPase-mediated degradation. Structural alterations in the 3′UTR result in increased aggR transcript stability, leading to increased AggR levels. We therefore investigated the effect of increased AggR levels on EAEC virulence. Upon finding the previously described AggR-dependent virulence factors, we detected novel AggR-regulated genes that may play relevant roles in EAEC virulence. Mutants exhibiting high AggR levels because of structural alterations in the aggR 3′UTR show increased mobility and increased pAA2 conjugation frequency. Furthermore, among the genes exhibiting increased fold change values, we could identify those of metabolic pathways that promote increased degradation of arginine, fatty acids and gamma-aminobutyric acid (GABA), respectively. In this paper, we discuss how the AggR-dependent increase in specific metabolic pathways activity may contribute to EAEC virulence.

SYNERGISTIC IMMUNOMODULATORY ACTIVITY OF PROBIOTICS BIFIDOBACTERIUM ANIMALIS AND LACTOBACILLUS CASEI IN ENTEROAGGREGATIVE ESCHERICHIA COLI (EAEC)-INFECTED CACO-2 CELLS.

Enteroaggregative Escherichia coli (EAEC) is an E. coli pathotype that presents aggregative adhesion patterns on in vitro cultivated cells, mainly related to persistent diarrhea cases in children. EAEC virulence factors are important for host colonization and pathogeni-city. Intestinal epithelial cells (IECs) recognize pathogen-associated molecular patterns (PAMPs) and initiate an immune response. Several studies using in vivo and in vitro models emphasize the probiotic activity and immunomodulatory capacity of Lactobacillus and Bifidobacterium species. To evaluate the modulation of cytokine production by probiotics Bifidobacterium animalis and Lactobacillus casei in human intestinal Caco-2 cells exposed to different strains of EAEC. Caco-2 cells were incubated with EAEC strains in the presence or absence of probiotics. The production of cytokines IL-8, TNF-α, IL-1β and IL-10 was evaluated in the supernatants by a sandwich enzyme-linked immunosorbent assay (ELISA). Cytokine production did not change when uninfected and EAEC-infected Caco-2 cells were exposed to probiotics separately. All EAEC induced a significant increase in IL-8 production by Caco-2 cells, but the probiotics, even together, could not reduce its production. On the other hand, the synergic activity of probiotic strains significantly increased TNF-α production but decreased the basal production of IL-1ß. Also, probiotics induced a significant increase in the production of the anti-inflammatory cytokine IL-10 during EAEC infection. Our results reinforce the synergistic immunomodulatory activity of probiotics during EAEC infection.

Role of the YehD fimbriae in the virulence-associated properties of enteroaggregative Escherichia coli.

The interaction of enteroaggregative Escherichia coli (EAEC) strains with the colonic gut mucosa is characterized by the ability of the bacteria to form robust biofilms, to bind mucin, and induce a local inflammatory response. These events are mediated by a repertoire of five different aggregative adherence fimbriae variants (AAF/I-V) typically encoded on virulence plasmids. In this study, we report the production in EAEC strains of a new YehD fimbriae (YDF), which is encoded by the chromosomal gene cluster yehABCD, also present in most E. coli strains. Immuno-labelling of EAEC strain 042 with anti-AAF/II and anti-YDF antibodies demonstrated the presence of both AAF/II and YDF on the bacterial surface. We investigated the role of YDF in cell adherence, biofilm formation, colonization of spinach leaves, and induction of pro-inflammatory cytokines release. To this aim, we constructed yehD deletion mutants in different EAEC backgrounds (strains 17-2, 042, 55989, C1010, 278-1, J7) each harbouring one of the five AAFs. The effect of the YDF mutation was strain dependent and AAF independent as the lack of YDF had a different impact on the phenotypes manifested by the different EAECs tested. Expression of the yehABCD operon in a E. coli K12 ORN172 showed that YDF is important for biofilm formation but not for adherence to HeLa cells. Lastly, screening of pro-inflammatory cytokines in supernatants of Caco-2 cells infected with EAEC strains 042 and J7 and their isogenic ΔyehD mutants showed that these mutants were significantly defective in release of IL-8 and TNF-α. This study contributes to the understanding of the complex and diverse mechanisms of adherence of EAEC strains and identifies a new potential target for preventive measures of gastrointestinal illness caused by EAEC and other E. coli pathogroups.

In silico molecular docking and in vitro antimicrobial efficacy of phytochemicals against multi-drug-resistant enteroaggregative Escherichia coli and non-typhoidal Salmonella spp.

BackgroundIn the wake of emergence of antimicrobial resistance, bioactive phytochemical compounds are proving to be important therapeutic agents. The present study envisaged in silico molecular docking as well as in vitro antimicrobial efficacy screening of identified phytochemical ligands to the dispersin (aap) and outer membrane osmoporin (OmpC) domains of enteroaggregative Escherichia coli (EAEC) and non-typhoidal Salmonella spp. (NTS), respectively.Materials and methodsThe evaluation of drug-likeness, molecular properties, and bioactivity of the identified phytocompounds (thymol, carvacrol, and cinnamaldehyde) was carried out using Swiss ADME, while Protox-II and StopTox servers were used to identify its toxicity. The in silico molecular docking of the phytochemical ligands with the protein motifs of dispersin (PDB ID: 2jvu) and outer membrane osmoporin (PDB ID: 3uu2) were carried out using AutoDock v.4.20. Further, the antimicrobial efficacy of these compounds against multi-drug resistant EAEC and NTS strains was determined by estimating the minimum inhibitory concentrations and minimum bactericidal concentrations. Subsequently, these phytochemicals were subjected to their safety (sheep and human erythrocytic haemolysis) as well as stability (cationic salts, and pH) assays.ResultsAll the three identified phytochemicals ligands were found to be zero violators of Lipinski’s rule of five and exhibited drug-likeness. The compounds tested were categorized as toxicity class-4 by Protox-II and were found to be non- cardiotoxic by StopTox. The docking studies employing 3D model of dispersin and ompC motifs with the identified phytochemical ligands exhibited good binding affinity. The identified phytochemical compounds were observed to be comparatively stable at different conditions (cationic salts, and pH); however, a concentration-dependent increase in the haemolytic assay was observed against sheep as well as human erythrocytes.ConclusionsIn silico molecular docking studies provided useful insights to understand the interaction of phytochemical ligands with protein motifs of pathogen and should be used routinely before the wet screening of any phytochemicals for their antibacterial, stability, and safety aspects.

Presumptive probiotic bacteria from traditionally fermented African food challenge the adhesion of enteroaggregative E. coli

AbstractColonization of intestinal tract with the potential to exclude, displace, and inhibit enteric pathogens is principally dependent on the adhesion ability of probiotics. Therefore, probiotic efficacy is considered to be mainly determined by their adhesion ability. The current study reports the antagonistic effect of four lactic acid bacteria (LAB) on the adhesion profile of four diarrhoeagenic and one non‐diarrhoeagenic enteroaggregative Escherichia coli (EAEC). All the bacterial strains investigated adhered to the Caco‐2 cells. All the LAB tested competitively excluded, displaced, and inhibited at least three (non‐) diarrhoeagenic EAEC strains from adhesion (p &lt; 0.05). In all, Lactobacillus plantarum, FS2 exhibited the strongest adhesion to the Caco‐2 cells, competitive exclusion (CE), displacement, and inhibition against most of the EAEC strains. Additionally, the competence to exclude, displace, and inhibit the EAEC from adhesion depended on both the pathogens and the LAB strains tested; signifying the participation of several mechanisms. Contrary to all the EAEC strains, gastro‐intestinal stress factors such as low pH (2.5) had no effect on the adhesion of the LAB. Unlike the gastro‐intestinal acidic conditions, bile salt conditioning (at pH 6.5) had no effect on the adhesion of both EAEC and LAB. In conclusion, all the LAB tested showed specific anti‐adherence effects including CE, displacement, and inhibition against the selected EAEC. The results indicate that all the LAB, particularly, the L. plantarum, FS2 had a good ability for exerting antagonistic effects against the selected EAEC for the prevention of gastrointestinal infection.

Prevalence, Virulence Gene Profiling, and Characterization of Enteroaggregative Escherichia coli from Children with Acute Diarrhea, Asymptomatic Nourished, and Malnourished Children Younger Than 5 Years of Age in India

To study the significance of enteroaggregative Escherichia coli (EAEC) as a pathogen causing acute diarrhea and a commensal in healthy nourished and malnourished children younger than five years of age in the Chandigarh region and to address possible traits of EAEC virulence genes, biofilm formation, phylogroups, and antibiotic resistance that would be correlated with diarrhea or carriage. Stool samples were obtained from children with acute diarrhea (n=548), as well as nourished (n=550), and malnourished controls without diarrhea (n=110). E coli isolates were confirmed as EAEC by pCVD432 polymerase chain reaction. Multiplex polymerase chain reactions were used to identify 22 virulence-related genes and phylogeny. Antibiotic susceptibility, adherence, and biofilm-forming potential also were studied. Overall, 16.6% of children were malnourished. EAEC detection was greater among children with acute diarrhea (16%) than nourished (6%) and malnourished nondiarrheal controls (2.7%). We found an association of EAEC infections with age <2years (P=.0001) in the diarrheal group. Adhesive variants adhesion fimbriae IV and adhesion fimbriae II were significantly associated with diarrhea. The aggR and aar genes showed a positive and negative association with the severity of disease (P=.0004 and P=.0003). A high degree of multidrug resistance was found (73.8%) in the diarrheal group. Most EAEC strains from the diarrheal group belonged to B2 and D phylogroups, whereas strains from non-diarrheal groups, which belonged to phylogroup B1. EAEC is a significant contributor to childhood diarrhea, its presence as a commensal, and the significance of the association of various virulence factors among the EAEC isolated from diarrheal and non-diarrheal stools. These data reinforce the importance of aggR and aar as positive and negative regulators and the contribution of AAF/II and AAF/IV fimbria for the pathobiology of EAEC.

The Superior Adherence Phenotype of E. coli O104:H4 is Directly Mediated by the Aggregative Adherence Fimbriae Type I

ABSTRACT Whereas the O104:H4 enterohemorrhagic Escherichia coli (EHEC) outbreak strain from 2011 expresses aggregative adherence fimbriae of subtype I (AAF/I), its close relative, the O104:H4 enteroaggregative Escherichia coli (EAEC) strain 55989, encodes AAF of subtype III. Tight adherence mediated by AAF/I in combination with Shiga toxin 2 production has been suggested to result in the outbreak strain’s exceptional pathogenicity. Furthermore, the O104:H4 outbreak strain adheres significantly better to cultured epithelial cells than archetypal EAEC strains expressing different AAF subtypes. To test whether AAF/I expression is associated with the different virulence phenotypes of the outbreak strain, we heterologously expressed AAF subtypes I, III, IV, and V in an AAF-negative EAEC 55989 mutant and compared AAF-mediated phenotypes, incl. autoaggregation, biofilm formation, as well as bacterial adherence to HEp-2 cells. We observed that the expression of all four AAF subtypes promoted bacterial autoaggregation, though with different kinetics. Disturbance of AAF interaction on the bacterial surface via addition of α-AAF antibodies impeded autoaggregation. Biofilm formation was enhanced upon heterologous expression of AAF variants and inversely correlated with the autoaggregation phenotype. Co-cultivation of bacteria expressing different AAF subtypes resulted in mixed bacterial aggregates. Interestingly, bacteria expressing AAF/I formed the largest bacterial clusters on HEp-2 cells, indicating a stronger host cell adherence similar to the EHEC O104:H4 outbreak strain. Our findings show that, compared to the closely related O104:H4 EAEC strain 55989, not only the acquisition of the Shiga toxin phage, but also the acquisition of the AAF/I subtype might have contributed to the increased EHEC O104:H4 pathogenicity.

Antimicrobial resistance and gene regulation in Enteroaggregative Escherichia coli from Egyptian children with diarrhoea: Similarities and differences

ABSTRACT Enteroaggregative Escherichia coli (EAEC) is a common diarrhoeagenic human pathogen, isolated from patients in both developing and industrialized countries, that is becoming increasingly resistant to many frontline antibiotics. In this study, we screened 50 E. coli strains from children presenting with diarrhea at the outpatients clinic of Assiut University Children’s Hospital, Egypt. We show that all of these isolates were resistant to multiple classes of antibiotics and identified two as being typical EAEC strains. Using whole genome sequencing, we determined that both isolates carried, amongst others, bla CTX-M and bla TEM antibiotic resistance genes, as well as many classical EAEC virulence determinants, including the transcriptional regulator, AggR. We demonstrate that the expression of these virulence determinants is dependent on AggR, including aar, which encodes for a repressor of AggR, Aar. Since biofilm formation is the hallmark of EAEC infection, we examined the effect of Aar overexpression on both biofilm formation and AggR-dependent gene expression. We show that whilst Aar has a minimal effect on AggR-dependent transcription it is able to completely disrupt biofilm formation, suggesting that Aar affects these two processes differently. Taken together, our results suggest a model for the induction of virulence gene expression in EAEC that may explain the ubiquity of EAEC in both sick and healthy individuals.

Comparative analysis of virulence determinants, phylogroups, and antibiotic susceptibility patterns of typical versus atypical Enteroaggregative E. coli in India.

Enteroaggregative Escherichia coli (EAEC) is an evolving enteric pathogen that causes acute and chronic diarrhea in developed and industrialized nations in children. EAEC epidemiology and the importance of atypical EAEC (aEAEC) isolation in childhood diarrhea are not well documented in the Indian setting. A comparative analysis was undertaken to evaluate virulence, phylogeny, and antibiotic sensitivity among typical tEAEC versus aEAEC. A total of 171 EAEC isolates were extracted from a broad surveillance sample of diarrheal (N = 1210) and healthy children (N = 550) across North India. Polymerase chain reaction (PCR) for the aggR gene (master regulator gene) was conducted to differentiate tEAEC and aEAEC. For 21 virulence genes, we used multiplex PCR to classify possible virulence factors among these strains. Phylogenetic classes were identified by a multiplex PCR for chuA, yjaA, and a cryptic DNA fragment, TspE4C2. Antibiotic susceptibility was conducted by the disc diffusion method as per CLSI guidelines. EAEC was associated with moderate to severe diarrhea in children. The prevalence of EAEC infection (11.4%) was higher than any other DEC group (p = 0.002). tEAEC occurrence in the diarrheal group was higher than in the control group (p = 0.0001). tEAEC strain harbored more virulence genes than aEAEC. astA, aap, and aggR genes were most frequently found in the EAEC from the diarrheal population. Within tEAEC, this gene combination was present in more than 50% of strains. Also, 75.8% of EAEC strains were multidrug-resistant (MDR). Phylogroup D (43.9%) and B1 (39.4%) were most prevalent in the diarrheal and control group, respectively. Genetic analysis revealed EAEC variability; the comparison of tEAEC and aEAEC allowed us to better understand the EAEC virulence repertoire. Further microbiological and epidemiological research is required to examine the pathogenicity of not only typical but also atypical EAEC.

Redefining enteroaggregative Escherichia coli (EAEC): Genomic characterization of epidemiological EAEC strains.

Although enteroaggregative E. coli (EAEC) has been implicated as a common cause of diarrhea in multiple settings, neither its essential genomic nature nor its role as an enteric pathogen are fully understood. The current definition of this pathotype requires demonstration of cellular adherence; a working molecular definition encompasses E. coli which do not harbor the heat-stable or heat-labile toxins of enterotoxigenic E. coli (ETEC) and harbor the genes aaiC, aggR, and/or aatA. In an effort to improve the definition of this pathotype, we report the most definitive characterization of the pan-genome of EAEC to date, applying comparative genomics and functional characterization on a collection of 97 EAEC strains isolated in the course of a multicenter case-control diarrhea study (Global Enteric Multi-Center Study, GEMS). Genomic analysis revealed that the EAEC strains mapped to all phylogenomic groups of E. coli. Circa 70% of strains harbored one of the five described AAF variants; there were no additional AAF variants identified, and strains that lacked an identifiable AAF generally did not have an otherwise complete AggR regulon. An exception was strains that harbored an ETEC colonization factor (CF) CS22, like AAF a member of the chaperone-usher family of adhesins, but not phylogenetically related to the AAF family. Of all genes scored, sepA yielded the strongest association with diarrhea (P = 0.002) followed by the increased serum survival gene, iss (p = 0.026), and the outer membrane protease gene ompT (p = 0.046). Notably, the EAEC genomes harbored several genes characteristically associated with other E. coli pathotypes. Our data suggest that a molecular definition of EAEC could comprise E. coli strains harboring AggR and a complete AAF(I-V) or CS22 gene cluster. Further, it is possible that strains meeting this definition could be both enteric bacteria and urinary/systemic pathogens.