Enteroaggregative Escherichia Coli Research Articles

Genome sequence of pan drug-resistant enteroaggregative Escherichia coli belonging to ST38 clone from India, an emerging EAEC/UPEC hybrid pathotype

Here, we report the genomic characterization of a pan drug-resistant (PDR) enteroaggregative Escherichia coli (EAEC) isolated from an immunocompromised infant who had diarrhea. The isolate belonged to the sequence type (ST) 38, which is a known enteroaggregative Escherichia coli (EAEC)/uropathogenic Escherichia coli (UPEC) hybrid strain having multi-drug resistance (MDR). The strain carried genes encoding multiple resistances to carbapenems, third-generation cephalosporins, polymyxin, fluoroquinolones, aminoglycosides, fosfomycin, nitrofurantoin, sulphonamides, and multiple efflux pump genes. Interspecies horizontal transfer, inter-strain, and clonal spread of these resistances to commensals and pathogens will be worrisome. We are concerned about the spread of such PDR strains. The genomic characterization of such strains will be useful in understanding the genetic makeup of EAEC/UPEC hybrid strains and developing new vaccines/diagnostics and therapeutics.

RpoS acts as a global repressor of virulence gene expression in E. coli O104:H4 and enteroaggregative E. coli.

In 2011, in Germany, Escherichia coli O104:H4 caused the enterohemorrhagic E. coli (EHEC) outbreak with the highest incidence rate of hemolytic uremic syndrome. This pathogen carries an exceptionally potent combination of EHEC- and enteroaggregative E. coli (EAEC)-specific virulence factors. Here, we identified an E. coli O104:H4 isolate that carried a single nucleotide polymorphism (SNP) in the start codon (ATG > ATA) of rpoS, encoding the alternative sigma factor S. The rpoS ATG > ATA SNP was associated with enhanced EAEC-specific virulence gene expression. Deletion of rpoS in E. coli O104:H4 Δstx2 and typical EAEC resulted in a similar effect. Both rpoS ATG > ATA and ΔrpoS strains exhibited stronger virulence-related phenotypes in comparison to wild type. Using promoter-reporter gene fusions, we demonstrated that wild-type RpoS repressed aggR, encoding the main regulator of EAEC virulence. In summary, our work demonstrates that RpoS acts as a global repressor of E. coli O104:H4 virulence, primarily through an AggR-dependent mechanism.

Characteristics of Pathogenic Escherichia coli Associated with Diarrhea in Children under Five Years in Northwestern Ethiopia.

Diarrheagenic Escherichia coli (DEC) are the leading cause of infectious diarrhea and pose a significant global, regional, and national burden of disease. This study aimed to investigate the prevalence of six DEC pathotypes in children with diarrhea and determine their antibiotic resistance patterns. Samples from 107 diarrheagenic children were collected and processed for Escherichia coli (E. coli). Single-plex PCR was used to detect target virulence genes as well as characterize and categorize DEC pathotypes. Antibiotic resistance patterns were determined by the Kirby-Bauer disk diffusion method. E. coli was detected in 79 diarrheal stool samples, accounting for 73.8% of the samples collected. Additionally, 49.4% (39 out of 79) of the isolates harbored various typical virulence factors. Results revealed six pathotypes of virulence: enterotoxigenic E. coli (ETEC) (53.8%), enteropathogenic E. coli (EPEC) (12.8%), enteroaggregative E. coli (EAEC) (10.3%), Heteropathotypes (7.8%), Shiga toxin-producing E. coli (STEC), and enterohemorrhagic E. coli (EHEC) (7.7% each). The isolates exhibited high antibiotic resistance against trimethoprim/sulfamethoxazole (82.1%), amoxicillin (79.5%), ampicillin (74.4%), gentamicin (69.2%), and streptomycin (64.1%). An overall occurrence of 84.6% of multiple-drug resistance was observed in the isolates, with resistance ranging from three to four antibiotic classes. Our findings revealed a high level of pathogenic E. coli that were highly resistant to multiple categories of antibiotics among children in the Awi zone. These findings highlight the potential role of pathogenic E. coli in childhood diarrhea in tropical low-resource settings and underscore the need for continued research on the characteristics of pathogenic and antibiotic-resistant strains.

Characterization of diarrheagenic Escherichia coli from different cattle production systems in Brazil

Diarrheagenic E. coli (DEC) can cause severe diarrhea and is a public health concern worldwide. Cattle are an important reservoir for this group of pathogens, and once introduced into the abattoir environment, these microorganisms can contaminate consumer products. This study aimed to characterize the distribution of DEC [Shiga toxin-producing E. coli (STEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), and enteroaggregative E. coli (EAEC)] from extensive and intensive cattle production systems in Brazil. Samples (n = 919) were collected from animal feces (n = 200), carcasses (n = 600), meat cuts (n = 90), employee feces (n = 9), and slaughterhouse water (n = 20). Virulence genes were detected by PCR in 10% of animal samples (94/919), with STEC (n = 81) as the higher prevalence, followed by EIEC (n = 8), and lastly EPEC (n = 5). Animals raised in an extensive system had a higher prevalence of STEC (average 48%, sd = 2.04) when compared to animals raised in an intensive system (23%, sd = 1.95) (Chi-square test, P < 0.001). From these animals, most STEC isolates only harbored stx2 (58%), and 7% were STEC LEE-positive isolates that were further identified as O157:H7. This study provides further evidence that cattle are potential sources of DEC, especially STEC, and that potentially pathogenic E. coli isolates are widely distributed in feces and carcasses during the slaughter process.

Colonization of the gut mucosa of colorectal cancer patients by pathogenic mucosa-associated Escherichia coli strains

Some strains of Escherichia coli are known to be involved in the pathogenesis of colorectal cancer (CRC). The aim of current study was to compare the general characteristics of the E. coli from CRC patients and healthy participants. A total of 96 biopsy samples from 48 CRC patients and 48 healthy participants, were studied. The clonality of the E. coli isolates was analyzed by Enterobacterial repetitive intergenic consensus-based PCR (ERIC-PCR) method. The strains were tested by PCR to determine the prevalence of different virulence factors. According to the results of ERIC-PCR analysis, (from the 860 E. coli isolates) 60 strains from CRC patients and 41 strains from healthy controls were identified. Interestingly, the majority of the strains of both groups were in the same cluster. Enteropathogenic E. coli (EPEC) was detected significantly more often in CRC patients (21.6 %) than in healthy participants (2.4 %) (p < 0.05). The Enteroaggregative E. coli (EAEC) was found in 18.33 % of the strains of CRC patients. However, other pathotypes were not found in the E. coli strains of both groups. Furthermore, all the studied genes encoding for virulence factors seemed to be more prevalent in the strains belonging to CRC patients. Among the virulence genes, the statistical difference regarding the frequency of fuyA, chuA, vat, papC, hlyA and cnf1 genes was found significant (p < 0.05). In conclusion, E. coli strains that carry extraintestinal pathogenic E. coli (ExPEC) and diarrheagenic E. coli (DEC) multiple virulence factors colonize the gut mucosa of CRC patients.

Enteric bacterial agents associated with diarrhea and their antimicrobial sensitivity profiles in children under 5 years from mukuru informal settlement, Nairobi, Kenya

BackgroundIn Kenya, diarrhoeal disease is the third leading cause of child mortality after malaria and pneumonia, accounting for nearly 100 deaths daily. We conducted a cross-sectional study in Mukuru informal settlements to determine the bacteria associated with diarrhea and their ASTs to provide data essential for implementing appropriate intervention measures.MethodsDiarrheagenic children (≤ 5 years) were purposively recruited from outpatient clinics of Municipal City Council, Mukuru kwa Reuben, Medical Missionaries of Mary, and Mama Lucy Kibaki Hospital, Nairobi. A total of 219 stool samples were collected between May 2021 and August 2021. Stool culture was done on MacConkey and Salmonella Shigella agar, while the recovered bacteria were identified using VITEK®2GNID and polymerase chain reaction (PCR) used for E. coli pathotyping. Antibiotic Susceptibility Testing was done using VITEK®2AST-GN83.ResultsAt least one bacterial organism was recovered from each of the 213 (97%) participants, with 115 (56%) participants having only one bacterial type isolated, 90 (43%) with two types of bacteria, and 2 (1%) with three types of bacteria recovered. The most predominant bacteria recovered was 85% (93/109) non-pathogenic E.coli and 15% (16/109)of pathogenic E.coli, with 2 (1%) were Enterohemorrhagic E.coli (EHEC), 6 (3%) were Enteroaggregative E.coli (EAEC), and 8 (4%) were Enteropathogenic E.coli (EPEC). Other potentially pathogenic bacteria included Enterobacter sp (27.8%), Klebsiella sp 33(11%), and Citrobacter sp 15(4.7%). Pathogenic isolates such as Salmonella 7 (2%), Proteus mirabilis 16 (6%), Providencia alcalifaciens 1 (0.3%), and Shigella 16 (4.7%) were detected. Isolates such as Pantoea spp 2(0.67%), Raoultella planticola 1(0.33%), and Kluyvera 6(2%) rarely reported but implicated with opportunistic diarrhoeal disease were also recovered. Ampicillin, cefazolin, and sulfamethoxazole-trimethoprim were the least effective antimicrobials at 64%, 57%, and 55% resistance, respectively, while meropenem (99%), amikacin (99%), tazobactam piperacillin (96%), and cefepime (95%) were the most effective. Overall, 33(21%) of all enterics recovered were multidrug-resistant.ConclusionThe study documented different bacteria potentially implicated with childhood diarrhea that were not limited to E. coli, Shigella, and Salmonella, as previously observed in Kenya. The strains were resistant to the commonly used antibiotics, thus narrowing the treatment options for diarrheal disease.

Plasmid-encoded toxin of Escherichia coli cleaves complement system proteins and inhibits complement-mediated lysis in vitro.

Plasmid-encoded toxin (Pet) is an autotransporter protein of the serine protease autotransporters of Enterobacteriaceae (SPATE) family, important in the pathogenicity of Escherichia coli. The pet gene was initially found in the enteroaggregative E. coli (EAEC) virulence plasmid, pAA2. Although this virulence factor was initially described in EAEC, an intestinal E. coli pathotype, pet may also be present in other pathotypes, including extraintestinal pathogenic strains (ExPEC). The complement system is an important defense mechanism of the immune system that can be activated by invading pathogens. Proteases produced by pathogenic bacteria, such as SPATEs, have proteolytic activity and can cleave components of the complement system, promoting bacterial resistance to human serum. Considering these factors, the proteolytic activity of Pet and its role in evading the complement system were investigated. Proteolytic assays were performed by incubating purified components of the complement system with Pet and Pet S260I (a catalytic site mutant) proteins. Pet, but not Pet S260I, could cleave C3, C5 and C9 components, and also inhibited the natural formation of C9 polymers. Furthermore, a dose-dependent inhibition of ZnCl2-induced C9 polymerization in vitro was observed. E. coli DH5α survived incubation with human serum pre-treated with Pet. Therefore, Pet can potentially interfere with the alternative and the terminal pathways of the complement system. In addition, by cleaving C9, Pet may inhibit membrane attack complex (MAC) formation on the bacterial outer membrane. Thus, our data are suggestive of a role of Pet in resistance of E. coli to human serum.

LACTIC ACID BACTERIA AND FERMENTED MAIZE SUPERNATANT (Omidun) HAVE ANTI-BIOFILM PROPERTIES AGAINST STAPHYLOCOCCI AND ENTEROAGGREGATIVE Escherichia coli STRAINS

Bacterial infections caused by biofilm forming organisms are of public health concern due to their propensity to contribute to persistent chronic diseases, chiefly because of their ability to resist antibiotics and host immune functions. Probiotics are considered useful therapeutic option in combating pathogenic biofilms. This study evaluates the anti-biofilm properties of potential probiotic Lactic Acid Bacteria (LAB) and fermented maize supernatant (Omidun) against selected biofilm-forming pathogens. Crystal violet biofilm assay was used to determine LAB and Omidun biofilm inhibition and dispersion in selected pathogens (Pseudomonas aeruginosa, Coagulase-negative staphylococci (CoNS), S. aureu and Enteroaggregative Escherichia coli) at different concentration (1%, 10%, 50%, 100%) of neutralized and non-neutralized cell free supernatant (CFS). Percentage biofilm inhibitions and dispersions were evaluated, and data were analysed with ANOVA. Omidun and LAB showed promising biofilm inhibitory and dispersive effect against the selected pathogens. L. plantarum showed the greatest biofilm inhibitory effect (P. aeruginosa: 7.85%, CoNS: 27.75%, S. aureus: 66.90%, EAEC: 39.73%) and dispersive effect (P. aeruginosa: 15.94%, CoNS: 23.27%, S. aureus: 24.90%, EAEC: 32.09%) against the selected pathogens while Omidun showed the least biofilm inhibitory and dispersive effect against the selected pathogens. There was no significance difference in the percentage of biofilm inhibition and dispersion produced under different concentrations, neutralized and non-neutralized state. Pseudomonas aeruginosa was the most resistant pathogen while Enteroaggregative Escherichia coli (EAEC) was the most susceptible. Inhibition and dispersion of biofilm can be mediated by LAB and Omidun, these effects appear to be independent of the produced organic acids

Complete genome sequences of a Canadian strain of enteroaggregative Escherichia coli (EAEC) with multiple metals and antimicrobial resistance genes isolated from municipal waste-activated sludge.

Enteroaggregative Escherichia coli (EAEC) is an emerging food-borne pathogen causing acute or persistent diarrhea in humans. Here, we report the complete genome sequence of a strain of EAEC with multiple metals and antimicrobial resistance genes isolated from a waste-activated sludge collected from a Canadian municipal wastewater treatment plant.

Enteroaggregative Escherichia coli in mid-Norway: A prospective, case control study.

The use of molecular methods has led to increased detection of Enteroaggregative Escherichia coli (EAEC) in faecal samples. Studies have yielded conflicting results regarding the clinical relevance of this finding. The objective of this study was to investigate the prevalence of EAEC in faecal samples from patients with diarrhoea and healthy controls and describe characteristics of EAEC positive persons. From March 1st, 2017 to February 28th, 2019, we investigated all consecutive faecal samples from patients with diarrhoea received at the laboratory and collected faecal samples from randomly invited healthy controls from mid-Norway. Real-time multiplex PCR was used for detection of bacterial, viral, and parasitic pathogens. We registered sex, age, urban versus non-urban residency, and travel history for all participants. Statistical analyses were performed with Pearson chi-squared test, Kruskal-Wallis test, and Mann-Whitney U test. We identified EAEC in 440 of 9487 (4.6%) patients with diarrhoea and 8 of 375 (2.2%) healthy controls. The EAEC prevalence was 19.1% among those with diarrhoea and recent foreign travel and 2.2% in those without travel history independent of diarrhoea. Concomitant pathogens were detected in 64.3% of EAEC-positive patients with diarrhoea. The median age was 28.5 in those with EAEC-positive diarrhoea and 38 in those with EAEC-negative diarrhoea (p <0.01). In patients with diarrhoea, travel was reported in 72% of those with EAEC and concomitant pathogens, and 54% and 12% in those with only EAEC and no EAEC, respectively (p <0.01). EAEC was a common detection, particularly in patients with diarrhoea and recent international travel, and was found together with other intestinal pathogens in the majority of cases. Our results suggest that domestically acquired EAEC is not associated with diarrhoea. Patients with EAEC-positive diarrhoea and concomitant pathogens were young and often reported recent travel history compared to other patients with diarrhoea.

Aksaray İli Buzağı İshallerinde Escherichia coli ve Escherichia coli Bakterisinin Son Üç Aylık Antibiyogram Sonuçları

Escherichia coli is a gram-negative, facultative anaerobic, motile, non-spore-forming rod-shaped bacterium belonging to the Enterobacteriaceae family. Pathogenic E. coli are divided into two groups: extraintestinal and intestinal. Intestinal Escherichia coli pathotypes: enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), Vero- or Shiga-toxin-producing E. coli (VTEC or STEC), enterohemorrhagic E. coli (EHEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC) and diffusely adherent E. coli (DAEC). Extraintestinal pathogenic E. coli: These can be listed as septicemic pathogenic E. coli, uropathogenic E. coli, avian pathogenic E. coli, breast pathogenic E. coli and those that cause uterine infections, endometrial pathogenic E. coli. Enterotoxigenic Escherichia coli (ETEC) is the most common cause of neonatal diarrhea in calves and is a bacterial disease that colonizes the small intestine, produces enterotoxin, and occurs among calves during the neonatal period. The aim of this study is to determine the E.coli isolates that cause neonatal calf diarrhea in the Aksaray region between January, February and March 2021. To be used in the study, internal organ samples (liver, heart, lung and mesenteric lymph node) of neonatal calves that died due to diarrhea were taken from 20 different cattle farms in the Aksaray region. One calf that died in each farm was included in the sampling, and a total of 20 calves were sampled. E.coli was isolated in 12 (60%) of the internal organ samples covering these three months. According to the antibiogram results of the samples, 50% of the isolates were converted to Amoxicillin and Erythromycin, 33.3% to Tetracycline, 58.3% to Trimethoprim-sulfamethoxazole, 66.6% to Streptomycin, 75% to Flofenicol, Gentamicin and Enrofloxacin, % 83.3 of them were found to be sensitive to Cefloxacin and Cefloxacinium.

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.

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

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

Biological characteristics of the enteroaggregative &lt;i&gt;escherichia coli &lt;/i&gt;ont:h30 18-726 (no. B-8857) strain isolated from a patient with ulcerative colitis and a new method of pcr identification

The aim of the work is to assess biological properties of the enteroaggregativeEscherichia coli(EAgEC) strain isolated from the colon mucosa biopsy material from a patient with ulcerative colitis, and to develop a new method for PCR identification ofE. colistrains.&#x0D; Materials and methods.The study involved 46 patients with a verified ulcerative colitis. Isolation of 87E. colistrains was carried out by using colon biopsy material obtained during a standard endoscopic procedure. The description of the biochemical bacterial properties was combined with the molecular genetic detection of virulence determinants and presence of antibiotic resistance mechanisms. Using the “AmpliSense®Escherichiose — FL” reagent kit, a screening for the presence of diarrheagenicE. colistrains was performed, which revealed the presence of a single strain of EAgEC. The methodological approach to creating a new method for strain identification was based on the search for a unique genetic sequence within the glutamate decarboxylase(gad)gene.&#x0D; Results.E. coli18-726 can be described biochemically typical to its species. The studied strain was characterized by a multiple resistance phenotype (MDR) to antibiotics, as well as a lack of sensitivity to five bacteriophages. TheE. coli18-726 strain had a unique sequence of the gene encoding the O-antigen, which differed from 188 known O-antigens (ONT) and, by the identity of the nucleotide sequence of the gene encoding the synthesis of the H-antigen, the strain belonged to the H30 serovar. Thus, the antigenic formula of strain EAgEC 18-726 was expressed as ONT:H30. TheE. coli18-726 strain contained a significant spectrum of genes that enable properties of virulence(iss, capU, aggA, aggB, aggC, aggD, aap, aar)and resistance to antibiotics(blaCTX-M-15, blaTEM-1B, aadA1, aadA5, mph(A), catB3). A new method for identifying EAgEC in chronic bowel disease is based on the polymerase chain reaction method using primers for a specific region of bacterial glutamate decarboxylase(gad)gene.Conclusion.1) The biochemical, antigenic and molecular genetic properties ofE. coliONT:H30 18-726 strain (No. B-8857) isolated from a patient with histomorphologically diagnosed ulcerative colitis were analyzed. 2) A method for PCR identification of EAgEC strains based on the detection of a specific region within the bacterial genomic glutamate decarboxylase gene has been created and patented.

Molecular mechanism of Enteroaggregative Escherichia coli induced apoptosis in cultured human intestinal epithelial cells.

Background. Enteroaggregative Escherichia coli (EAEC) is an evolving etiological agent of acute and persistent diarrhoea worldwide. The previous study from our laboratory has reported the apoptosis-inducing activity of EAEC in human small intestinal and colonic epithelial cell lines. In the present investigation, we have explored the underlying mechanism of EAEC-induced apoptosis in human intestinal epithelial cell lines.Methods. INT-407 and HCT-15 cells were infected with EAEC-T8 and EAEC-pT8 (plasmid cured strain of EAEC-T8) separately. Cells cultured in the absence of bacteria served as a negative control in all the experiments. For the subsequent experiments, the molecular mechanism(s) of epithelial cell aposptosis was measured in EAEC infecting both the cell lines by flow cytometry, real-time PCR and Western blotting.Results and conclusions. EAEC was found to activate the intrinsic/mitochondrial apoptotic pathway in both the cell lines through upregulation of pro-apoptotic Bax and Bak, un-alteration/reduction in the level of anti-apoptotic Bcl-2 and Bcl-XL, decrease in mitochondrial transmembrane potential, accumulation of cytosolic cytochrome c leading to activation of procaspase-9 and procaspase-3, which ultimately resulted in DNA fragmentation and apoptosis. Further, an increased expression of Fas, activation of procaspase-8 and upregulation of pro-apoptotic Bid in the EAEC-infected cells indicated the involvement of extrinsic apoptotic pathway too in this process. Our finding has undoubtedly led to an increased understanding of EAEC pathogenesis, which may be helpful to develop an improved strategy to combat the infection.

The tip protein PAAR is required for the function of the type VI secretion system.

Bacteria are constantly competing to colonize crowded ecological niches, such as the human gut. The type VI secretion system (T6SS) is a critical bacterial weapon in this warfare. It resembles a crossbow with a poisoned arrow allowing bacteria to inject toxic effectors directly into target cells. This machinery is formed by an envelope-spanning complex which recruits the baseplate, an assembly platform allowing the polymerization of a contractile structure. The tail consists of a tube surrounded by a sheath and topped by the needle complex composed of the VgrG and PAAR proteins. In the enteric pathogen enteroaggregative Escherichia coli (EAEC), the Tle1 phospholipase toxin ensures the antibacterial activity of the T6SS-1. For its transport, Tle1 interacts directly with the trimeric spike protein VgrG. However, the importance and the function of the tip protein PAAR in the T6SS remain unclear. Here, we characterized the PAAR protein of EAEC using biochemical, fluorescence microscopy, and antibacterial competition approaches. Using pull-down assays and cryo-electron microscopy analysis of the (VgrG)3-(Tle1)3-PAAR complex, we show that PAAR tops and closes the β-prism structure of the VgrG spike. The PAAR protein structure is further tightened by the zinc atom coordinated via conserved residues essential for its function. We provide evidence that PAAR is necessary for T6SS-1-mediated killing due to its requirement for proper T6SS baseplate assembly and further sheath polymerization. Our results suggest that the PAAR protein is an essential component of T6SS. IMPORTANCE The type VI secretion system (T6SS) is a bacterial contractile injection system involved in bacterial competition by the delivery of antibacterial toxins. The T6SS consists of an envelope-spanning complex that recruits the baseplate, allowing the polymerization of a contractile tail structure. The tail is a tube wrapped by a sheath and topped by the tip of the system, the VgrG spike/PAAR complex. Effectors loaded onto the puncturing tip or into the tube are propelled in the target cells upon sheath contraction. The PAAR protein tips and sharpens the VgrG spike. However, the importance and the function of this protein remain unclear. Here, we provide evidence for association of PAAR at the tip of the VgrG spike. We also found that the PAAR protein is a T6SS critical component required for baseplate and sheath assembly.

Study of Escherichia coli as a Cause of Diarrhoea in the Ashanti Region of Ghana

Even though diarrhoeagenic Escherichia coli (E. Coli) is accepted worldwide as essential diarrhoea-causing agent, especially among children, routinely they are not sought as a stool sample pathogen in many microbiological laboratories. The conventional biochemical means are not able to differentiate E. coli which is a normal flora in the gastrointestinal tract from the pathogenic strains. This research work sought to detect and identify diarrhoeagenic E.coli in the Ashanti region of Ghana by using conventional multiplex PCR. A convenient sampling method was employed to select a total of 502 patients from Ashanti Regional and St. Michael’s Hospitals for the study. In all, five pathotypes of diarrhoeagenic E.coli were detected with enterotoxigenic E. coli (ETEC) as the most frequent. The target genes considered for each group of diarrhoeagenic E. coli were eaeA (E. coli attaching- effacing) and bfpA (bundle forming pilus A) for enteropathogenic E. coli (EPEC), elt and Stla for enterotoxigenic E. coli (ETEC), ial for enteroinvasive E. coli (EIEC), CVD432 for enteroaggregative E. coli (EAEC), and hlyA for enterohaemorrhagic E. coli (EHEC). Generally, some of the participants (31.08% = n/N= 69/222) exhibited symptoms of diarrhoea while others did not (68.92% = n/N =153/222 ), although they were positive for the pathotypes. The virulence factors considered were seen as contributory factors to the symptomatic situations. Participants in the ≤5 and  ≥ 42-year groups were seen to be more vulnerable to diarrhoea. Additionally, it was discovered that men were more susceptible to diarrhoea than women. The study therefore recommends more studies into the ≥ 42-year group not neglecting that of children. In addition, this current study suggests the routine utilization of molecular methods such as conventional multiplex PCR to identify and detect the pathotypes of E. coli causing diarrhoea.

Analysing the impact of eriosematin E from Eriosema chinense Vogel. against different diarrhoeagenic pathovars of Escherichia coli using in silico and in vitro approach

Since diarrhoea is reportedly the third largest cause of fatality among kids, therefore it is considered to be one of the major areas of concerns among developing nations. The main causative agents of diarrhoea include Escherichia coli, Vibrio cholera, and Shigella spp where E. coli shares the maximum contribution. The roots of the plant Eriosema chinense Vogel. (Fabaceae) are traditionally used by the native tribes of Meghalaya, India to treat diarrhoea. From previous reports, the plant and its marker eriosematin E have been reported to have antidiarrhoeal potential against pathogenic and nonpathogenic diarrhoea. Therefore, the objective of the current investigation was to use in silico studies to determine the efficacy of eriosematin E against different diarrhoeagenic strains of E. coli. Six different pathovars of E. coli i.e. enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enterohaemorrhagic E. coli (EHEC), enteroaggregative E. coli (EAEC), uropathogenic E. coli (UPEC) and enteroinvasive E. coli (EIEC) were subjected to docking simulation studies utilizing Glide module of Schrodinger Maestro 2018-1 MM Share Version. Based on the obtained binding energy and balance between H-bonding, hydrophobic, and salt bridge interactions eriosematin E was found to be most effective against EPEC followed by EAEC and ETEC, while UPEC and EHEC were moderately affected. The molecular dynamics studies suggested a higher affinity of eriosematin E towards heat-labile enterotoxin b-pentamer from ETEC. The in vitro antibacterial studies against the universal strain S. aureus 12981 and E. coli 10418 revealed the effectiveness of eriosematin E showing MIC values of ≥256 µg/mL. Communicated by Ramaswamy H. Sarma

PCR diagnostics are insufficient for the detection of Diarrhoeagenic Escherichia coli in Ibadan, Nigeria.

Understanding the contribution of different diarrhoeagenic Escherichia coli pathotypes to disease burden is critical to mapping risk and informing vaccine development. Targeting select virulence genes by PCR is the diagnostic approach of choice in high-burden, least-resourced African settings. We compared the performance of a commonly-used multiplex protocol to whole genome sequencing (WGS). PCR was applied to 3,815 E. coli isolates from 120 children with diarrhoea and 357 healthy controls. Three or more isolates per specimen were also Illumina-sequenced. Following quality assurance, ARIBA and Virulencefinder database were used to identify virulence targets. Root cause analysis of deviant PCR results was performed by examining target sensitivity using BLAST, Sanger sequencing false-positive amplicons, and identifying lineages prone to false-positivity using in-silico multilocus sequence typing and a Single Nucleotide Polymorphism phylogeny constructed using IQTree. The sensitivity and positive predictive value of PCR compared to WGS ranged from 0-77.8% while specificity ranged from 74.5-94.7% for different pathotypes. WGS identified more enteroaggregative E. coli (EAEC), fewer enterotoxigenic E. coli (ETEC) and none of the Shiga toxin-producing E. coli detected by PCR, painting a considerably different epidemiological picture. Use of the CVD432 target resulted in EAEC under-detection, and enteropathogenic E. coli eae primers mismatched more recently described intimin alleles common in our setting. False positive ETEC were over-represented among West Africa-predominant ST8746 complex strains. PCR precision varies with pathogen genome so primers optimized for use in one part of the world may have noticeably lower sensitivity and specificity in settings where different pathogen lineages predominate.

[Drug resistance and molecular typing of diarrheagenic escherichia coli from the diarrhea clinics in Qingpu District, Shanghai City from 2015 to 2019].

Objective: To analyze the drug resistance and multilocus sequence typing of five types of diarrheagenic Escherichia coli (DEC) isolated from diarrhea outpatients of diarrhea comprehensive monitoring designated hospital in Qingpu District, Shanghai City from 2015 to 2019. Methods: From January 2015 to December 2019, five types of DEC, isolated and identified from diarrhea outpatient cases' anal swabs of the Qingpu branch of Zhongshan Hospital were collected to determine the minimal inhibitory concentration by using the micro broth dilution susceptibility test. The strains, resistant to the third-generation cephalosporins or carbapenems, or producing ESBLs, were selected based on the results of sensitivity tests and determined by WGS. The MLST typing of DEC was analyzed based on the WGS technology and the minimum spanning tree was constructed by BioNumerics 7.6 software to analyze the local dominant flora. Results: A total of 513 strains of DEC were detected and isolated from 4 494 anal swabs, with a detection rate of 11.42%. About 500 strains were tested for drug sensitivity to nine antibiotics in four classes, including 330 strains of enterotoxigenic E.coli (ETEC), 72 strains of enteroaggregative E.coli (EAEC), 95 strains of enteropathogenic E.coli (EPEC), 1 strain of enterohemorrhagic E.coli (EHEC), and 2 strains of enteroinvasive E.coli (EIEC). From 2015 to 2019, the resistance rate of cefotaxime-clavulanic acid was significantly different (P<0.05). The resistance rate of virulence types of DEC to nalixic acid was significantly different (P<0.05). About 71 strains of DEC were determined by WGS, and 77 drug-resistant genes were detected. Strains were classified into 32 ST subtypes, with the dominant genotypes being ST-1491 (29.6%, 21/71) and ST-10 Complex (23.9%, 17/71). All ST-1491 produced ESBLs, which were blaCTX-M gene mutant strains. The dominant type of ST-10 complex was ST-218 (35.3%, 6/17). In addition, 8 strains of EAEC, 14 strains of EPEC and 49 strains of ETEC were classified into 7, 14 and 18 ST subtypes, respectively. Conclusion: The drug resistance of DEC strains from the diarrhea outpatient case of Qingpu District is serious. The ST types of EAEC and EPEC are highly polymorphic. The dominant ST types of DEC are basically consistent with the common genotypes in southeast China.