Enteroaggregative Escherichia Coli Research Articles

Enteroaggregative Escherichia coli induced activation of epidermal growth factor receptor contributes to IL-8 secretion by cultured human intestinal epithelial cells

Enteroaggregative Escherichia coli (EAEC) has been identified as a new enteropathogen that causes acute and chronic diarrhea in children and travelers. One defining aspect of EAEC-pathogenesis is the induction of an inflammatory response in intestinal epithelium. In this study, we have found that EAEC-induced EGFR activation in human small intestinal and colonic epithelial was attenuated in the presence of a specific inhibitor of EGFR (Tyrphostin AG1478). Further, the aggregative stacked-brick type of adherence of this organism to both the cell lines and this pathogen-induced cytoskeletal rearrangement of these cells was also reduced in the presence of Tyrphostin AG1478. Moreover, EAEC-induced activation of downstream effectors (ERK-1/2, PI3K and Akt) of EGFR mediated cell signaling pathways were found to be suppressed in the presence of EGFR inhibitor. A decrease in IL-8 response in EAEC infected both the cell types were also noted in the presence of specific inhibitors of these downstream effectors, transcription factors and Tyrphostin AG1478. We propose that EAEC-induced activation of EGFR is quintessential for stacked-brick adherence of EAEC to human intestinal epithelial cells, their cytoskeletal rearrangements and stimulation of ERK-1/2 and PI3K/Akt mediated signal transduction pathways, resulting in the activation of NF-κB, AP-1, STAT-3 and finally IL-8 secretion by these cells.

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.

Highly-conserved regulatory activity of the ANR family in the virulence of diarrheagenic bacteria through interaction with master and global regulators

ANR (AraC negative regulators) are a novel class of small regulatory proteins commonly found in enteric pathogens. Aar (AggR-activated regulator), the best-characterized member of the ANR family, regulates the master transcriptional regulator of virulence AggR and the global regulator HNS in enteroaggregative Escherichia coli (EAEC) by protein–protein interactions. On the other hand, Rnr (RegA-negative regulator) is an ANR homolog identified in attaching and effacing (AE) pathogens, including Citrobacter rodentium and enteropathogenic Escherichia coli (EPEC), sharing only 25% identity with Aar. We previously found that C. rodentium lacking Rnr exhibits prolonged shedding and increased gut colonization in mice compared to the parental strain. To gain mechanistic insights into this phenomenon, we characterized the regulatory role of Rnr in the virulence of prototype EPEC strain E2348/69 by genetic, biochemical, and human organoid-based approaches. Accordingly, RNA-seq analysis revealed more than 500 genes differentially regulated by Rnr, including the type-3 secretion system (T3SS). The abundance of EspA and EspB in whole cells and bacterial supernatants confirmed the negative regulatory activity of Rnr on T3SS effectors. We found that besides HNS and Ler, twenty-six other transcriptional regulators were also under Rnr control. Most importantly, the deletion of aar in EAEC or rnr in EPEC increases the adherence of these pathogens to human intestinal organoids. In contrast, the overexpression of ANR drastically reduces bacterial adherence and the formation of AE lesions in the intestine. Our study suggests a conserved regulatory mechanism and a central role of ANR in modulating intestinal colonization by these enteropathogens despite the fact that EAEC and EPEC evolved with utterly different virulence programs.

Phenotypic and genotypic characterization of pathogenic Escherichia coli identified in resistance mapping of β-lactam drug-resistant isolates from seafood along Tuticorin coast.

The ubiquity of pathogenic E. coli isolate possessing antimicrobial resistance was investigated in seafood samples procured from major seafood supply chain markets established for export and domestic consumption along Tuticorin coast. Out of 63 seafood samples examined, 29 (46%) were found to be contaminated by pathogenic E. coli harbouring one or more genes of virulent potential. Based on virulome profiling, 9.55% of isolates belonged to enterotoxigenic E. coli (ETEC), 8.08% to enteroaggregative E. coli (EAEC), 7.35% to enterohemorrhagic E. coli (EHEC), 2.20% to enteropathogenic E. coli (EPEC), and 2.20% to uropathogenic E. coli (UPEC). All the 34 virulome positive and haemolytic pathogenic E. coli have been serogrouped as O119, O76, O18, O134, O149, O120, O114, O25, O55, O127, O6, O78, O83, O17 and clinically significant O111, O121, O84, O26, O103, and O104 (non-O157 STEC) serotypes in this study. Multi-drug resistance (MDR) (≥ 3 antibiotic classes/sub-classes) was exhibited in 38.23% of the pathogenic E. coli, and 17.64% were extensive drug resistant (XDR). Extended spectrum of β-lactamase (ESBL) genotypes were confirmed in 32.35% isolates and 20.63% isolates harboured ampC gene. One sample (Penaeus semisulcatus) collected from landing centre (L1) harboured all ESBL genotypes blaCTX-M, blaSHV, blaTEM, and ampC genes. Hierarchical clustering of isolates revealed the separation of ESBL isolates into three clusters and non-ESBL isolates into three clusters based on phenotypic and genotypic variations. Based on dendrogram analysis on antibiotic efficacy pattern, carbapenems and β-lactam inhibitor drugs are the best available treatment for ESBL and non-ESBL infections. This study emphasizes the significance of comprehensive surveillance of pathogenic E. coli serogroups that pose serious threat to public health and compliance of AMR antimicrobial resistant genes in seafood that hinder seafood supply chain.

Epidemiology of Enteroaggregative, Enteropathogenic, and Shiga Toxin–ProducingEscherichia coliAmong Children Aged <5 Years in 3 Countries in Africa, 2015–2018: Vaccine Impact on Diarrhea in Africa (VIDA) Study

To address knowledge gaps regarding diarrheagenic Escherichia coli (DEC) in Africa, we assessed the clinical and epidemiological features of enteroaggregative E. coli (EAEC), enteropathogenic E. coli (EPEC), and Shiga toxin-producing E. coli (STEC) positive children with moderate-to-severe diarrhea (MSD) in Mali, The Gambia, and Kenya. Between May 2015 and July 2018, children aged 0-59 months with medically attended MSD and matched controls without diarrhea were enrolled. Stools were tested conventionally using culture and multiplex polymerase chain reaction (PCR), and by quantitative PCR (qPCR). We assessed DEC detection by site, age, clinical characteristics, and enteric coinfection. Among 4840 children with MSD and 6213 matched controls enrolled, 4836 cases and 1 control per case were tested using qPCR. Of the DEC detected with TAC, 61.1% were EAEC, 25.3% atypical EPEC (aEPEC), 22.4% typical EPEC (tEPEC), and 7.2% STEC. Detection was higher in controls than in MSD cases for EAEC (63.9% vs 58.3%, P < .01), aEPEC (27.3% vs 23.3%, P < .01), and STEC (9.3% vs 5.1%, P < .01). EAEC and tEPEC were more frequent in children aged <23 months, aEPEC was similar across age strata, and STEC increased with age. No association between nutritional status at follow-up and DEC pathotypes was found. DEC coinfection with Shigella/enteroinvasive E. coli was more common among cases (P < .01). No significant association was detected between EAEC, tEPEC, aEPEC, or STEC and MSD using either conventional assay or TAC. Genomic analysis may provide a better definition of the virulence factors associated with diarrheal disease.

Antibiofilm Effect of Biogenic Silver Nanoparticles Combined with Oregano Derivatives against Carbapenem-Resistant Klebsiella pneumoniae.

Resistant bacteria may kill more people than COVID-19, so the development of new antibacterials is essential, especially against microbial biofilms that are reservoirs of resistant cells. Silver nanoparticles (bioAgNP), biogenically synthesized using Fusarium oxysporum, combined with oregano derivatives, present a strategic antibacterial mechanism and prevent the emergence of resistance against planktonic microorganisms. Antibiofilm activity of four binary combinations was tested against enteroaggregative Escherichia coli (EAEC) and Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC): oregano essential oil (OEO) plus bioAgNP, carvacrol (Car) plus bioAgNP, thymol (Thy) plus bioAgNP, and Car plus Thy. The antibiofilm effect was accessed using crystal violet, MTT, scanning electron microscopy, and Chromobacterium violaceum anti-quorum-sensing assays. All binary combinations acted against preformed biofilm and prevented its formation; they showed improved antibiofilm activity compared to antimicrobials individually by reducing sessile minimal inhibitory concentration up to 87.5% or further decreasing biofilm metabolic activity and total biomass. Thy plus bioAgNP extensively inhibited the growth of biofilm in polystyrene and glass surfaces, disrupted three-dimensional biofilm structure, and quorum-sensing inhibition may be involved in its antibiofilm activity. For the first time, it is shown that bioAgNP combined with oregano has antibiofilm effect against bacteria for which antimicrobials are urgently needed, such as KPC.

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.

Genomic characterization of endemic diarrheagenic Escherichia coli and Escherichia albertii from infants with diarrhea in Vietnam.

Diarrheagenic Escherichia coli (DEC) is a group of bacterial pathogens that causes life-threatening diarrhea in children in developing countries. However, there is limited information on the characteristics of DEC isolated from patients in these countries. A detailed genomic analysis of 61 DEC-like isolates from infants with diarrhea was performed to clarify and share the characteristics of DEC prevalent in Vietnam. DEC was classified into 57 strains, including 33 enteroaggregative E. coli (EAEC) (54.1%), 20 enteropathogenic E. coli (EPEC) (32.8%), two enteroinvasive E. coli (EIEC) (3.3%), one enterotoxigenic E. coli (ETEC), and one ETEC/EIEC hybrid (1.6% each), and surprisingly into four Escherichia albertii strains (6.6%). Furthermore, several epidemic DEC clones showed an uncommon combination of pathotypes and serotypes, such as EAEC Og130:Hg27, EAEC OgGp9:Hg18, EAEC OgX13:H27, EPEC OgGp7:Hg16, and E. albertii EAOg1:HgUT. Genomic analysis also revealed the presence of various genes and mutations associated with antibiotic resistance in many isolates. Strains that demonstrate potential resistance to ciprofloxacin and ceftriaxone, drugs recommended for treating childhood diarrhea, accounted for 65.6% and 41%, respectively. Our finding indicate that the routine use of these antibiotics has selected resistant DECs, resulting in a situation where these drugs do not provide in therapeutic effects for some patients. Bridging this gap requires continuous investigations and information sharing regarding the type and distribution of endemic DEC and E. albertii and their antibiotic resistance in different countries.

Nursery outbreak caused by enteroaggregative Escherichia coli serogroup O111:H21.

This study describes, for the first time, the occurrence of an epidemic of enteroaggregative Escherichia coli (EAEC) O111:H21 in a Belgian nursery and describes a practical approach concerning its management. Few data exist in the literature on this type of outbreak. Clinical and microbiological investigations were needed to find a link between the cases and identify the causative agent. The microbiological procedure followed was first based on conventional analyses: isolation using selective cultures, identification by MALDI-TOF MS, antibiogram, determination of the serogroup by agglutination, then whole genome sequencing. A total of 7/21 children were infected with this pathogen. Four cases could be confirmed by a molecular technique, wgMLST, as belonging to the same bacterial clone. The action plan put in place focused on symptomatic case eviction, strict general hygiene precaution as well as specific cleaning and disinfection measures. The epidemic did last only a few days. It appears important, in the context of an epidemic, that clinical laboratories standardize their practice by equipping themselves with molecular techniques such as a multiplex which does not focus only on the serotype O157:H7 and which make it possible to distinguish the different pathotypes of E. coli by targeting several virulence genes (stx, aggR…). However, cost/effectiveness studies are awaited to confirm the interest of a systematic search by molecular method for the pathogen involved in a suspected outbreak occurring in a nursery.

Antibiotic resistance, pathotypes, and pathogen-host interactions in Escherichia coli from hospital wastewater in Bulawayo, Zimbabwe.

This study aimed to characterise E. coli strains isolated from hospital wastewater effluent in Bulawayo, Zimbabwe, using both molecular and cytological approaches. Wastewater samples were aseptically collected from the sewerage mains of a major public referral hospital in Bulawayo province weekly for one month. A total of 94 isolates were isolated and confirmed as E. coli through biotyping and PCR targeting the uidA housekeeping gene. A total of 7 genes (eagg, eaeA, stx, flicH7, ipaH, lt, and st genes) coding for virulence in diarrheagenic E. coli were targeted. Antibiotic susceptibility of E. coli was determined against a panel of 12 antibiotics through the disk diffusion assay. The infectivity status of the observed pathotypes was investigated using HeLa cells through adherence, invasion, and intracellular assay. None of the 94 isolates tested positive for the ipaH and flicH7genes. However, 48 (53.3%) isolates were enterotoxigenic E. coli (ETEC) (lt gene positive), 2 (2.13%) isolates were enteroaggregative E. coli (EAEC) (eagg gene), and 1 (1.06%) isolate was enterohaemorrhagic E. coli (EHEC) (stx and eaeA). A high level of sensitivity was observed in E. coli against ertapenem (98.9%), and Azithromycin (75.5%). The highest resistance was against ampicillin (92.6%) and sulphamethoxazole-trimethoprim (90.4%). Seventy-nine (84%) E. coli isolates exhibited multidrug resistance. The infectivity study results indicated that environmentally isolated pathotypes were as infective as the clinically isolated pathotypes for all three parameters. No adherent cells were observed using ETEC, and no cells were observed in the intracellular survival assay using EAEC. This study revealed that hospital wastewater is a hotspot for pathogenic E. coli and that the environmentally isolated pathotypes maintained their ability to colonise and infect mammalian cells.

Detection and antibiotic resistance of diarrheagenic Escherichia coli from patients with diarrhea in Ulaanbaatar, Mongolia.

Diarrheal diseases are common with worldwide distribution, and diarrheagenic Escherichia coli (DEC) strains are the main causative agents. The present study aimed to define the association of various pathotypes of E. coli from diarrheal patients in Mongolia. A total of 341 E. coli strains were isolated from the stool of diarrheal patients. Bacterial susceptibility to antimicrobial agents was determined by the Kirby Bauer disk diffusion method. DEC isolates were identified by HEp-2 cell adherence assay and multiplex polymerase chain reaction (PCR). DEC pathogens were detected in 53.7% of 341 E. coli isolates. Enteroaggregative E. coli (EAEC) was the most common DEC pathotype identified by HEp-2 adherence assay and multiplex PCR methods in 97 samples (28.4%), followed by atypical enteropathogenic E. coli (EPEC) in 50 samples (14.7%), diffusely adherent E. coli (DAEC) in 25 samples (7.3%), enterohaemorrhagic E. coli (EHEC) in 6 samples (1.8%), enterotoxigenic E. coli (ETEC) in 4 samples (1.2%), and enteroinvasive E. coli (EIEC) in 1 sample (0.3%). DEC strains had > 50% antibiotic resistance against cephalothin, ampicillin, and trimethoprim/sulfamethoxazole. All tested DEC strains were susceptible to imipenem. Among the 183 DEC strains, 27 (14.8%) were extended spectrum beta-lactamase producing isolates, and 125 (68.3%) isolates were multiple drug resistant. We have identified six pathotypes of DEC from the clinical isolates tested and concluded that a high prevalence of antimicrobial resistance was observed in these pathotypes. EAEC was the most common pathotype identified and this is the first report of EHEC identification in Mongolia.

Occurrence of diarrheagenic Escherichia coli pathotypes from raw milk and unpasteurized buttermilk by culture and multiplex polymerase chain reaction in southwest Iran.

In developing countries including Iran, there are limited data on diarrheagenic Escherichia coli (DEC) contamination in milk and unpasteurized buttermilks. This study aimed to determine the occurrence of DEC pathotypes by culture and multiplex polymerase chain reaction (M-PCR) in some dairy products from southwest Iran. In this cross-sectional study (September to October 2021), 197 samples (87 unpasteurized buttermilk and 110 raw cow milk) were collected from dairy stores of Ahvaz, southwest Iran. The presumptive E. coli isolates were primarily identified using biochemical tests and then confirmed by PCR of uidA gene. The occurrence of 5 DEC pathotypes: enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), and enteroinvasive E. coli (EIEC) were investigated using M-PCR. Overall, 76 (76/197, 38.6%) presumptive E. coli isolates were identified by biochemical tests. Using uidA gene, only 50 isolates (50/76, 65.8%) were confirmed as E. coli. DEC pathotypes were detected in 27 of 50 (54.0%) E. coli isolates (74.1%, 20/27 from raw cow milk and 25.9%, 7/27 from unpasteurized buttermilk). The frequency of DEC pathotypes was as follows: 1 (3.7%) EAEC, 2 (7.4%) EHEC, 4 (14.8%) EPEC, 6 (22.2%) ETEC, and 14 (51.9%) EIEC. However, 23 (46.0%) E. coli isolates had only the uidA gene and were not considered DEC pathotypes. Possible health risks for Iranian consumers can be attributed to the presence of DEC pathotypes in dairy products. Hence, serious control and prevention efforts are needed to stop the spread of these pathogens.

Evaluación de la diarrea crónica en pacientes con reciente diagnóstico de infección por VIH mediante el panel gastrointestinal FilmArray®

IntroductionThe treatment and diagnosis of chronic diarrhea in the immunocompromised patient depends on the ability to rapidly detect the etiologic agents. AimsOur aim was to evaluate the results of the FilmArray® gastrointestinal panel in patients newly diagnosed with HIV infection that presented with chronic diarrhea. Material and methodsUtilizing nonprobability consecutive convenience sampling, 24 patients were included that underwent molecular testing for the simultaneous detection of 22 pathogens. ResultsIn 24 HIV-infected patients with chronic diarrhea, enteropathogen bacteria were detected in 69% of the cases, parasites in 18%, and viruses in 13%. Enteropathogenic Escherichiacoli and enteroaggregative Escherichiacoli were the main bacteria identified, Giardia lamblia was found in 25%, and norovirus was the most frequent viral agent. The median number of infectious agents per patient was three (range of 0 to 7). The biologic agents not identified through the FilmArray® method were tuberculosis and fungi. ConclusionsSeveral infectious agents were simultaneously detected through the FilmArray® gastrointestinal panel in patients with HIV infection and chronic diarrhea.

Isolation and characterization of enteroaggregative Escherichia coli among the causes of bacterial diarrhea in children

Diarrhea is a public health problem and an important cause of morbidity and mortality, enteroaggregative Escherichia coli (EAEC) have been recognized increasingly as agents of diarrhea in developing and in industrial countries. So the aim of this study is to study the prevalence of EAEC, its virulence factors and study it´s resistance to antibiotics. Results showed that E. coli represent 86 (32.9%) of the isolated bacteria. the high percentage 60% were EAEC, plasmid-encoded toxin (pet) was the marker most prevalent detected 14/30(28%) than EAST1(for EaggEC heat-stable enterotoxin 1)which detected in 9/30(18%) of EAEC and two virulence genes (pet+ and EAST1+) were found in7/30(14 %) of EAEC. 10% of isolates were contained capsule but only 3.3%isolates of E. coli have Type Ι pili while all the isolates possess Type ΙΙΙ pili. The results in the present study indicated that all E. coli were negative to Congo red dye agar test in contrast high percent of E. coli isolates were able to produce biofilm (86.7%) with different degree of thickness, while 20%isolates were urease positive after 24- 48 hrs, and 10% isolates were able to produce amylase. All isolates of E. coli were unable to produce Lecithinase, nuclease and hemolysin. While, 93.3% of E. coli isolates produced β-lactamase. The frequency of Sorbitol E. coli fermenter was 66.7%. In this study EAEC isolates were resistance (100%) to Penicillin and Ampicillin, High resistance(96.7%) was observed to each of Ceftriaxone, Cefotaxime and Trimethoprim. and Tetracycline (76.7%), resistance percentage of Both Amikacin and Rifampicin were (40%) and (33.3%) respectively. Higher sensitivity was reported to Gentamicin (63.3%), Ciprofloxacin(60%) then Nitrofurantoin (56.7%). The data from this study draw attention to the importance of notifying diarrheal disease. The high level of antimicrobial resistance observed in our study raises a broader discussion about the indiscriminate use or misuse of antibiotics and the risks of empirical antibiotic therapy in children of a very young age.

Role of ST6GAL1 and ST6GAL2 in subversion of cellular signaling during enteroaggregative Escherichia coli infection of human intestinal epithelial cell lines

Emerging evidence have suggested that aberrant sialylation on cell-surface carbohydrate architecture may influence host-pathogen interactions. The α2,6-sialyltransferase (ST) enzymes were found to alter the glycosylation pattern of the pathogen-infected host cell-surface proteins, which could facilitate its invasion. In this study, we assessed the role of specific α2,6-ST enzymes in the regulation of enteroaggregative E. coli (EAEC)-induced cell signaling pathways in human intestinal epithelial cells. EAEC-induced expression of α2,6-ST family genes in HCT-15 and INT-407 cell lines was assessed at mRNA level by qRT-PCR. Specific esi-RNA was used to silence the target ST-gene in each of the EAEC-infected cell type. Subsequently, the role of these enzymes in regulation of EAEC-induced cell signaling pathways was unraveled by analyzing the expression of MAPkinases (ERK1/2, p38, JNK) and transcription factors (NFκB, cJun, cFos, STAT) at mRNA and protein levels by qRT-PCR and western immunoblotting, respectively, expression of selected sialoglycoproteins by western immunoblotting along with the secretory IL-8 response using sandwich ELISA. ST6GAL-1 and ST6GAL-2 were efficiently silenced in EAEC-infected HCT-15 and INT-407 cells, respectively. Significant reduction in EAEC-induced activation of MAPKs, transcription factors, sialoglycoproteins, and IL-8 secretion was noted in ST-silenced cells in comparison to the respective control cells. We propose that ST6GAL-1 and ST6GAL-2 are quintessential for EAEC-induced stimulation of MAPK-mediated pathways, resulting in activation of transcription factors, leading to an inflammatory response in the human intestinal epithelial cells. Our study may be helpful to design better therapeutic strategies to control EAEC- infection. KEY POINTS: • EAEC induces α2,6-sialyltransferase (ST) upregulation in intestinal epithelial cells • Target STs (ST6GAL-1 & ST6GAL-2) were efficiently silenced using specific esiRNAs • Expression of MAPKs, transcription factors & IL-8 was reduced in ST silenced cells.

Impact of enteropathogens on faltering growth in a resource-limited setting.

Environmental enteropathy is an important contributor to childhood malnutrition in the developing world. Chronic exposure to fecal pathogens leads to alteration in intestinal structure and function, resulting in impaired gut immune function, malabsorption, and growth faltering leading to environmental enteropathy. A community-based intervention study was carried out on children till 24 months of age in Matiari district, Pakistan. Blood and fecal specimens were collected from the enrolled children aged 3-6 and 9 months. A real-time PCR-based TaqMan array card (TAC) was used to detect enteropathogens. Giardia, Campylobacter spp., enteroaggregative Escherichia coli (EAEC), Enteropathogenic Escherichia coli (EPEC), Enterotoxigenic Escherichia coli (ETEC), and Cryptosporidium spp. were the most prevailing enteropathogens in terms of overall positivity at both time points. Detection of protozoa at enrollment and 9 months was negatively correlated with rate of change in height-for-age Z (ΔHAZ) scores during the first and second years of life. A positive association was found between Giardia, fecal lipocalin (LCN), and alpha 1-Acid Glycoprotein (AGP), while Campylobacter spp. showed positive associations with neopterin (NEO) and myeloperoxidase (MPO). Protozoal colonization is associated with a decline in linear growth velocity during the first 2 years of life in children living in Environmental enteric dysfunction (EED) endemic settings. Mechanistic studies exploring the role of cumulative microbial colonization, their adaptations to undernutrition, and their influence on gut homeostasis are required to understand symptomatic enteropathogen-induced growth faltering.

Antibiotic sensitivity profile of bacterial isolates from stool samples among children below five years in Murang'a County, Kenya.

the discovery of antibiotics led to the optimistic belief of completely eradicating infectious diseases during the golden era following their discovery. Countries are grappling with the burden of microbial resistance bringing a near paralysis of all facets of mankind. Enterobacteriaceae and other hard-to-treat Gram-negative bacteria have become resistant to nearly all antibiotic options available, and this is a bad taste in the fight against microbial resistance. during the months of April-October 2017, 163 children below five years presenting with diarrhea were randomly selected in Murang´a and Muriranja´s Hospitals. Bacterial agents were identified and antibiotic susceptibility profile was determined. Design: a cross-sectional study approach was used. Statistical analyses were performed using STATA v. 13. a total of 188 bacteria belonging to 11 genera were isolated, and identified and their antibiotic susceptibility profiles were determined. Susceptibility testing showed that almost all the Enterotoxigenic Escherichia coli (ETEC), Enteropathogenic Escherichia coli (EPEC), Enteroaggregative Escherichia coli (EAEC), Salmonella, Klebsiella, Shigella, Vibrio, Enterobacter, Proteus, Pseudomonas, Aeromonas, Citrobacter and Yersinia species were resistant to the following antibiotics: ampicillin, amoxicillin, chloramphenicol, ciprofloxacin, ceftriaxone and kanamycin. Other than ETEC (90.9%), all the rest of the isolates were resistant to nalidixic acid. Other than ETEC (9.1%), EAEC (33.3%) and Salmonella species (95.2%), all the rest of the isolates were resistant to gentamicin. Other than V. cholerae, all the other isolates were resistant to trimethoprim-sulfamethoxazole. Isolates were sporadically resistant to erythromycin, streptomycin, doxycycline, and ofloxacin. the high resistance rate of enteric Gram-negative bacterial pathogens in Murang´a County is alarming. The need for urgent, efficient, and sustainable actions and interventions, such as culture and susceptibility testing, is needed and must be taken into account to minimize and prevent the establishment and spread of enteric pathogenic bacteria.

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.

Genome Assessment of Carbapenem- and Colistin-Resistant Escherichia coli from Patients in a Sentinel Hospital in China.

Antimicrobial-resistant (AMR) pathogens are a significant threat to public health worldwide. However, the primary carrier of AMR genes, particularly against last-resort antibiotics, is still only partially studied in Chinese hospitals. In a sentinel hospital in China, we collected 157 E. coli strains from patients between January and July 2021. One blaNDM-1-, nine blaNDM-5-, and one mcr-1-positive E. coli recovered from inpatients were identified as resistant to meropenem and colistin. There are 37 virulence genes discovered in the 11 strains, including astA in strain EC21Z-147 (O128: H4), which belongs to the enteroaggregative E. coli (EAEC). The blaNDM gene is distributed into distinct ST types, including ST48, ST616, ST410, ST711, and ST2003, while the mcr-1 gene was identified in ST117. The conjugative plasmids IncX3, IncI1-I, and IncI2 mediated the blaNDM-5 and mcr-1 genes detected among inpatients. Notably, the youngest age at which mcr-1-positive E. coli has been reported was at one day old, in a child in which the strain is closely related to strains with animal origins. Hospitals are major environments for the spread and dissemination of critical virulence and AMR genes, which requires active monitoring systems at the genome level to surveil the spread of virulence and AMR.