Enteroaggregative Escherichia Coli Isolates Research Articles

Enteroaggregative Escherichia coli (EAEC) isolates obtained from non-diarrheic children carry virulence factor-encoding genes from Extraintestinal Pathogenic E. Coli (ExPEC).

Enteroaggregative E. coli (EAEC) is one of the most frequent pathogens isolated from diarrheal patients as well as from healthy individuals in Brazil and has recently also been implicated as an extraintestinal pathogenic E. coli (ExPEC) associated with bloodstream and urinary tract infections. In this study, 37 EAEC isolates, obtained from fecal samples of non-diarrheic children, were molecularly and phenotypically characterized to access the pathogenic features of these isolates. The EAEC isolates were assigned into the phylogroups A (54.1%), D (29.7%), B1 (13.5%) and B2 (2.7%); and harbored genes responsible for encoding the major pilin subunit of the aggregative adherence fimbriae (AAFs) or aggregate-forming pili (AFP) adhesins as follows: aggA (24.3%), agg3A (5.4%), agg4A (27.0%), agg5A (32.4%) and afpA (10.8%). The most frequent O:H serotypes were O15:H2 (8.1%), O38:H25 (5.4%) and O86:H2 (5.4%). Twenty-one isolates (56.8%) produce the aggregative adherence (AA) pattern on HeLa cells, and biofilm formation was more efficient among EAEC isolates harboring the aggA and agg5A genes. PFGE analysis showed that 31 (83.8%) of the isolates were classified into 10 distinct clusters, which reinforces the high diversity found among the isolates studied. Of note, 40.5% (15/37) of the EAEC isolates have a genetic profile compatible with E. coli isolates with intrinsic potential to cause extraintestinal infections in healthy individuals, and therefore, classified as EAEC/ExPEC hybrids. In conclusion, we showed the presence of EAEC/ExPEC hybrids in the intestinal microbiota of non-diarrheic children, possibly representing the source of some endogenous extraintestinal infections.

Infection of mice by the enteroaggregative E. coli strain 042 and two mutant derivatives overexpressing virulence factors: impact on disease markers, gut microbiota and concentration of SCFAs in feces

Several pathogenic Escherichia coli strains cause diarrhea. Enteroaggregative E. coli (EAEC) strains are one of the diarrheagenic pathotypes. EAEC cells form a “stacked-brick” arrangement over the intestinal epithelial cells. EAEC isolates express, among other virulence determinants, the AggR transcriptional activator and the aggregative adherence fimbriae (AAF). Overexpression of the aggR gene results in increased expression of virulence factors such as the aff genes, as well as several genes involved in specific metabolic pathways such as fatty acid degradation (fad) and arginine degradation (ast). To support the hypothesis that induction of the expression of some of these pathways may play a role in EAEC virulence, in this study we used a murine infection model to evaluate the impact of the expression of these pathways on infection parameters. Mice infected with a mutant derivative of the EAEC strain 042, characterized by overexpression of the aggR gene, showed increased disease symptoms compared to those exhibited by mice infected with the wild type (wt) strain 042. Several of these symptoms were not increased when the infecting mutant, which overexpressed aggR, lacked the fad and ast pathways. Therefore, our results support the hypothesis that different metabolic pathways contribute to EAEC virulence.

Caracterización molecular de Escherichia coli diarreogénica proveniente de población pediátrica ambulatoria con diarrea, atendida en dos hospitales de Buenos Aires, Argentina

Diarrheagenic Escherichia coli comprises a heterogeneous group of pathotypes or pathogenic variants that share phenotypic characteristics with marked differences in virulence genes, colonization sites, pathogenesis, clinical presentation, and epidemiology of infection. The most studied pathotypes are Shiga toxin-producing E.coli (STEC), enterotoxigenic E.coli (ETEC), enteropathogenic E.coli (EPEC), enteroaggregative E.coli (EAEC), and enteroinvasive E.coli (EIEC). The objective of the study was to characterize the isolates of diarrheagenic E.coli from an outpatient pediatric population with diarrhea attended in two public hospitals from Buenos Aires, Argentina. Diarrheagenic E.coli pathotypes were investigated by amplifying characteristic virulence gene fragments: intimin (eae), heat-labile toxin (lt), heat-stable toxins (stp, sth), invasion plasmid antigen H (ipaH), transcriptional activator R (aggR) and Shiga toxins (stx1, stx2). Molecular subtyping of isolates was performed using PFGE (XbaI). Diarrheagenic E.coli was detected in 14% (84/601) of cases. The EAEC pathotype was prevalent, while ETEC, STEC, EPEC and EIEC were found in a lower proportion. EAEC isolates exhibited a high degree of genetic diversity. All pathotypes were found in children under 5years of age, while only EAEC, EIEC and ETEC were detected in the older population. Future studies that include the characterization of isolates from a greater number of genes and populations from other geographical areas will be necessary to determine the relevance of diarrheagenic E.coli in Argentina.

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.

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.

Molecular Epidemiology of Enteroaggregative Escherichia coli (EAEC) Isolates of Hospitalized Children from Bolivia Reveal High Heterogeneity and Multidrug-Resistance.

Enteroaggregative Escherichia coli (EAEC) is an emerging pathogen frequently associated with acute diarrhea in children and travelers to endemic regions. EAEC was found the most prevalent bacterial diarrheal pathogen from hospitalized Bolivian children less than five years of age with acute diarrhea from 2007 to 2010. Here, we further characterized the epidemiology of EAEC infection, virulence genes, and antimicrobial susceptibility of EAEC isolated from 414 diarrheal and 74 non-diarrheal cases. EAEC isolates were collected and subjected to a PCR-based virulence gene screening of seven virulence genes and a phenotypic resistance test to nine different antimicrobials. Our results showed that atypical EAEC (a-EAEC, AggR-negative) was significantly associated with diarrhea (OR, 1.62, 95% CI, 1.25 to 2.09, p < 0.001) in contrast to typical EAEC (t-EAEC, AggR-positive). EAEC infection was most prevalent among children between 7–12 months of age. The number of cases exhibited a biannual cycle with a major peak during the transition from warm to cold (April–June). Both typical and a-EAEC infections were graded as equally severe; however, t-EAEC harbored more virulence genes. aap, irp2 and pic were the most prevalent genes. Surprisingly, we detected 60% and 52.6% of multidrug resistance (MDR) EAEC among diarrheal and non-diarrheal cases. Resistance to ampicillin, sulfonamides, and tetracyclines was most common, being the corresponding antibiotics, the ones that are frequently used in Bolivia. Our work is the first study that provides comprehensive information on the high heterogenicity of virulence genes in t-EAEC and a- EAEC and the large prevalence of MDR EAEC in Bolivia.

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.

Genotypic and phenotypic characterization of enteroaggregative Escherichia coli (EAEC) isolates from diarrheic children: An unresolved diagnostic paradigm exists.

Objective(s):The enteroaggregative Escherichia coli (EAEC) has been one of the most intriguing emerging bacterial pathogens in children that occur both in developing countries and the industrial world. Although various phenotypic and genotypic based protocols have been suggested for diagnosis of EAEC, they are not conclusive or practical to be used in most clinical laboratories. Materials and Methods:In this study, we analyzed and compared 36 typical EAEC strains (aggR-positive) by various genotypic and phenotypic methods.Results:Briefly, pCVD432 was detected in all of isolates along with aggR, then it was followed by other virulence genes including app, astA, aggA, and pet genes in 32 (88.8%), 21 (58.3%), 9 (25%), and 2 (5.5%) isolates, respectively. Biofilm was formed by 34 (94.4%) isolates, while only 26 (72.2%) isolates showed an aggregative adherence pattern to HEp-2 cells. Conclusion:The genetic and phenotypic features of EAEC were highly inconsistent, which may have considerable diagnostic implications. The variations in the virulence genes, phenotypic characteristics, and genetic profiles among the EAEC isolates again emphasized the genetic heterogeneity of this emerging pathotype. Biofilm formation may be an important phenotypic virulence property of this pathotype, especially in strains with the aggR-pCVD432-aap-astA profile.

Genetic and Virulence Profiles of Enteroaggregative Escherichia coli (EAEC) Isolated From Deployed Military Personnel (DMP) With Travelers' Diarrhea.

To discern if there was a particular genotype associated with clinical enteroaggregative Escherichia coli (EAEC) strains isolated from deployed military personnel (DMP) with travelers' diarrhea (TD), we characterized a collection of EAEC from DMP deployed to Afghanistan, Djibouti, Kenya, or Honduras. Although we did not identify a specific EAEC genotype associated with TD in DMP, we found that EAEC isolated at the first clinic visit were more likely to encode the dispersin gene aap than EAEC collected at follow-up visits. A majority of the EAEC isolates were typical EAEC that adhered to HEp-2 cells, formed biofilms, and harbored genes for aggregative adherence fimbriae (AAF), AggR, and serine protease autotransporters of Enterobacteriaceae (SPATEs). A separate subset of the EAEC had aggR and genes for SPATEs but encoded a gene highly homologous to that for CS22, a fimbriae more commonly found in enterotoxigenic E. coli. None of these CS22-encoding EAEC formed biofilms in vitro or adhered to HEp-2 cells. Whole genome sequence and single nucleotide polymorphism analyses demonstrated that most of the strains were genetically diverse, but that a few were closely related. Isolation of these related strains occurred within days to more than a year apart, a finding that suggests a persistent source and genomic stability. In an ampicillin-treated mouse model we found that an agg4A+ aar- isolate formed a biofilm in the intestine and caused reduced weight gain in mice, whereas a strain that did not form an in vivo biofilm caused no morbidity. Our diverse strain collection from DMP displays the heterogeneity of EAEC strains isolated from human patients, and our mouse model of infection indicated the genotype agg4A+ aar– and/or capacity to form biofilm in vivo may correlate to disease severity.

Analysis of the Virulence Profile and Phenotypic Features of Typical and Atypical Enteroaggregative Escherichia coli (EAEC) Isolated From Diarrheal Patients in Brazil.

Enteroaggregative Escherichia coli (EAEC) is an important agent of acute and persistent diarrhea in children and adults worldwide. Here we report a characterization of 220 EAEC isolates, 88.2% (194/220) of which were typical and 11.8% (26/220) were atypical, obtained from diarrheal patients during seven years (2010-2016) of epidemiological surveillance in Brazil. The majority of the isolates were assigned to phylogroups A (44.1%, 97/220) or B1 (21.4%, 47/220). The aggregative adherence (AA) pattern was detected in 92.7% (204/220) of the isolates, with six of them exhibiting AA concomitantly with a chain-like adherence pattern; and agg5A and agg4A were the most common adhesin-encoding genes, which were equally detected in 14.5% (32/220) of the isolates. Each of 12 virulence factor-encoding genes (agg4A, agg5A, pic, aap, aaiA, aaiC, aaiG, orf3, aar, air, capU, and shf) were statistically associated with typical EAEC (P < 0.05). The genes encoding the newly described aggregate-forming pili (AFP) searched (afpB, afpD, afpP, and afpA2), and/or its regulator (afpR), were exclusively detected in atypical EAEC (57.7%, 15/26), and showed a significant association with this subgroup of EAEC (P < 0.001). In conclusion, we presented an extensive characterization of the EAEC circulating in the Brazilian settings and identified the afp genes as putative markers for increasing the efficiency of atypical EAEC diagnosis.

Myoviridae phage PDX kills enteroaggregative Escherichia coli without human microbiome dysbiosis.

Introduction Bacteriophage therapy can be developed to target emerging diarrhoeal pathogens, but doing so in the absence of microbiome disruption, which occurs with antibiotic treatment, has not been established.Aim Identify a therapeutic bacteriophage that kills diarrhoeagenic enteroaggregative Escherichia coli (EAEC) while leaving the human microbiome intact.Methodology Phages from wastewater in Portland, OR, USA were screened for bacteriolytic activity by overlay assay. One isolated phage, PDX, was classified by electron microscopy and genome sequencing. A mouse model of infection determined whether the phage was therapeutic against EAEC. 16S metagenomic analysis of anaerobic cultures determined whether a normal human microbiome was altered by treatment.Results Escherichia virus PDX, a member of the strictly lytic family Myoviridae, killed a case-associated EAEC isolate from a child in rural Tennessee in a dose-dependent manner, and killed EAEC isolates from Columbian children. A single dose of PDX (multiplicity of infection: 100) 1 day post-infection reduced EAEC recovered from mouse faeces. PDX also killed EAEC when cultured anaerobically in the presence of human faecal bacteria. While the addition of EAEC reduced the β-diversity of the human microbiota, that of the cultures with either faeces alone, faeces with EAEC and PDX, or with just PDX phage was not different statistically.Conclusion PDX killed EAEC isolate EN1E-0007 in vivo and in vitro, while not altering the diversity of normal human microbiota in anaerobic culture, and thus could be part of an effective therapy for children in developing countries and those suffering from EAEC-mediated traveller’s diarrhoea without causing dysbiosis.

Wide genetic heterogeneity and low antimicrobial resistance of enteroaggregative Escherichia coli isolates from several rural communities.

Enteroaggregative Escherichia coli (EAEC), a highly heterogeneous pathotype of E. coli classified as typical and atypical, are an emerging cause of acute and persistent diarrhea. We aimed to investigate whether population living in rural geographic areas, impacts in the heterogeneity, dissemination and antimicrobial susceptibility of EAEC strains. EAEC isolates (n=73) were analysed for the presence of 23 putative virulence factors, plasmid and antimicrobial resistance profiles, biofilm formation, pulsedfield gel electrophoresis (PFGE) and by multilocus sequence typing (MLST). The agg3A, agg4A, agn43, aap, shf, astA, pet, pic/set1A and sat genes, biofilm forming and antimicrobial resistance were statistically associated with typical EAEC. A low frequency of all isolates was resistant or showed a multidrug-resistance profile. No isolate showed the same plasmid profile. In total, 58 different pulsotypes were observed. Sixteen isolates analysed by MLST belonged to 15 different sequence types (ST) and showed a different PFGE pattern and virulence-gene profile. The fact that the communities are semi-isolated did not impact on the peculiar heterogeneity of EAEC, being characterized as epidemiologically independent strains.

Prevalence of Virulence Genes in Enteroaggregative Escherichia coli Isolates from Young Children from Rural South Africa.

In this study, we report on the prevalence of 19 virulence genes in enteroaggregative Escherichia coli (EAEC) isolates from northern South Africa. Stool samples obtained prospectively from 97 children from 1 to 12 months of age were analyzed, and EAEC isolates were confirmed based on the presence of aaiC or aatA genes. We investigated 177 enteroaggregative Escherichia coli isolates for the prevalence of virulence genes using multiplex polymerase chain reaction. The chromosomal gene aaiC was detected at higher frequency (48.0%) compared with aatA (26.0%). The gene encoding the open reading frame Orf61 was the most prevalent putative virulence trait detected among the isolates (150/177; 84.7%). None of the genes was statistically associated with diarrhea (P > 0.05). Detection rates were higher during 7-12 month of life with an association observed for the pic gene and the age group 7-12 months (P = 0.04). Winter was the season with the highest detection rates. Our data reveal a high prevalence of Orf61, Orf3, and astA in South African EAEC isolates. Specific genes may provide additional markers for the study of disease associations with age and season of sample collection.

Escherichia coli enterotoxigénica y enteroagregativa: prevalencia, patogénesis y modelos múridos.

Enterotoxigenic (ETEC) and enteroaggregative Escherichia coli (EAEC) pathotypes are important etiological agents causative of diarrhea in children younger than 5 years of age in Mexico and in developing countries, where they cause numerous deaths. Both have been associated with delayed growth in children and are the main causative agents of traveler's diarrhea. The pathogenesis of both bacteria starts by adhering to the intestinal epithelium by means of fimbriae, called colonization factors in human ETEC isolates and aggregative adherence fimbriae in EAEC isolates. Once ETEC adheres to the enterocyte, it produces one or both of its toxins and induces the secretion of chloride and sodium ions and water into the intestinal lumen, producing its characteristic watery diarrhea. EAEC binds to the intestinal epithelium forming a biofilm, induces the production of mucus, releases its toxins and promotes inflammation. EAEC and ETEC infection models with wild-type C57BL/6 and CD40 ligand-deficient mice (with intact microbiota), respectively, revealed that undernutrition and low-zinc diet increases EAEC infection, causing growth retardation, and that ETEC colonizes, persists and induces local and systemic humoral immune response.

Analysis of bacterial etiology of the outpatients with infectious diarrhea in the Children′s Hospital Capital Institute of Pediatrics

Objective To analyze the bacterial etiology and drug resistance of pathogens from the outpatients with acute infectious diarrhea in the Children′s Hospital Capital Institute of Pediatrics. Methods The children with acute infectious diarrhea in the outpatient department of our hospital were randomly enrolled in the period of August 2017 to July 2018. Enteric pathogenic bacteria were cultured and isolated from the fresh stool specimens. The serum of Salmonella spp were identified while five types of diarrheagenic Escherichia coli were detected by PCR method. Antibiotic susceptibility of Salmonella spp and diarrheagenic Escherichia coli were tested by Kirby-Bauer disk diffusion method.Chi-square or Fisher exact test was done for statistical analysis. Results Of the 356 stool specimens, 103 strains of 6 pathogenic bacterial species were detected. The positive rate was 27.8% (99/356), and 2 strains were detected in four stool samples. The isolation rate of Salmonella spp, which was the highest in this study, was 10.7% (38/356). The isolation rate of Staphylococcus aureus, diarrheagenic Escherichia coli and Pseudomonas aeruginosa were 8.4% (30/356), 5.6% (20/356) and 3.7% (13/356), respectively. No Shigella spp strain was isolated. Salmonella enteritidis and Salmonella typhimurium were the dominant serotype for 38 Salmonella spp stains with the isolation rate of 63.2% (24/38). Each 10 Enteropathogenic Escherichia coli and 10 Enteroaggregative Escherichia coli isolates were detected among the diarrheagenic Escherichia coli. Enterohemorrhagic Escherichia coli, Enteroinvasive Escherichia coli and Enterotoxigenic Escherichia colistrain was not found in this study. The resistant rates of Salmonella spp to ampicillin and ceftriaxone were 52.6% (20/38) and 13.2% (5/38), while the diarrheagenic Escherichia coli were 11/20 and 1/20, respectively. Three diarrheagenic Escherichia coli isolates produced extended spectrum beta-lactamase. Conclusions Salmonella spp was the major bacterial pathogen among the outpatients with infectious diarrhea in the hospital. The study indicated that the infection ratios of Staphylococcus aureus, diarrheagenic Escherichia coli and Pseudomonas aeruginosa increased. Shigella spp was no long the main pathogenic agent. The resistant rates of Salmonella spp and diarrheagenic Escherichia coli to ampicillin were high, but low resistant rates to ceftriaxone were found among these two kinds of bacteria. The third-generation cephalosporins should be the first choice for the treatment of childhood bacterial diarrhea. Key words: Diarrhea; Salmonella; Escherichia coli; Drug resistance,Bacterial; Child

Emergence of Resistance to Quinolones and β-Lactam Antibiotics in Enteroaggregative and Enterotoxigenic Escherichia coli Causing Traveler's Diarrhea.

The objective of this study was to assess the antimicrobial resistance of enteroaggregative Escherichia coli (EAEC) and enterotoxigenic E. coli (ETEC) strains causing traveler's diarrhea (TD) and to investigate the molecular characterization of antimicrobial resistance genes to third-generation cephalosporins, cephamycins, and quinolones. Overall, 39 EAEC and 43 ETEC clinical isolates were studied. The susceptibilities of EAEC and ETEC against ampicillin, amoxicillin-clavulanic acid, cefotaxime, imipenem, chloramphenicol, tetracycline, co-trimoxazole, nalidixic acid, ciprofloxacin, azithromycin, and rifaximin were determined. All genes encoding resistance determinants were detected by PCR or PCR plus DNA sequencing. The epidemiology of selected EAEC and ETEC strains was studied using multilocus sequence typing (MLST). The resistance to quinolones of EAEC and ETEC strains causing TD has significantly increased over the last decades, and high percentages have been found especially in patients traveling to India and sub-Saharan Africa. Sequence type 38 (ST38) and ST131, carrying the blaCTX-M-15 and blaCTX-M-27 genes, respectively, are highly prevalent among extended-spectrum β-lactamase (ESBL)-producing EAEC and ETEC strains. The cephamycinase ACT-20 is described in the present study for the first time in EAEC and ETEC strains causing TD in patients who had traveled to Central America. The percentages of resistance to azithromycin in EAEC and ETEC isolates from patients to Southeast Asia/India and Africa are above 25%. Meanwhile, rifaximin is still active against EAEC and ETEC, with the prevalence of resistant strains not being high. In conclusion, fluoroquinolones should no longer be considered the drugs of choice for the prevention or treatment in TD for travelers traveling to India and Africa. Azithromycin and rifaximin are still a good alternative to treat TD caused by EAEC or ETEC.

CHARACTERIZATION OF ENTEROAGGREGATIVE ESCHERICHIA COLI AMONG DIARRHEAL CHILDRENIN WESTERN BRAZILIAN AMAZON.

Enteroaggregative Escherichia coli (EAEC) is one of the main acute and chronic diarrhea causes both in children and adults, mainly in developing countries. The aim of the present study is to characterize EAEC strains isolated from faecal samples and to identify genes potentially contributing to virulence, biofilm production and antimicrobial resistance in children admitted to a pediatric hospital in Porto Velho, Rondônia State. The total of 1,625 E. coli specimens were isolated from 591 children in the age group 6 years or younger who were hospitalized in Cosme and Damião Children Hospital in Porto Velho, between February 2010 and February 2012, with acute gastroenteritis. Colonies suggestive of E. coli were subjected to polymerase chain reaction testing in order to identify the virulence factors. The in vitro adhesion assays using HEp-2 adherence were tests. Biofilm detection through spectrophotometry and antimicrobial susceptibility tests were conducted in the disk diffusion method. The mentioned study examined 591 stool samples from children with diarrhea. Diarrheogenic E. coli was found in 27.4% (162/591) of the children. EAEC was the diarreagenic E. coli most frequently associated with diarrhea 52.4% (85/162), which was followed by enteropathogenic E. coli 43.8% (71/162), enterotoxigenic E. coli 2.4% (4/162), and enterohemorrhagic E. coli 1.2% (2/162). The aggR gene was detected in 63.5% (54/85) of EAEC isolates; moreover, statistically significant correlation was observed among typical EAEC (aggR) and aatA (P<0.0001), irp2 (P=0.0357) and shf (P=0.0328). It was recorded that 69% (59/85) of the 85 analyzed EAEC strains were biofilm producers; 73% (43/59) of the biofilm producers carried the aggR gene versus 42.3% (11/26) of non-producers (P=0.0135). In addition, there was association between the aatA gene and biofilm production; 61% (36/59) of the samples presented producer strains, versus 19.2% (5/26) of non-producers (P<0.0004). Antibiotic sensitivity test evidenced that most EAEC were ampicillin 70.6% (60/85), sulfamethoxazole 60% (51/85), tetracycline 44.7% (38/85) and cefotaxime 22.4% (19/85) resistant. As far as it is known, the present study is pioneer in Northern Brazil to investigate EAEC virulence factors and to show the antimicrobial susceptibility of EAEC strains isolated from children with diarrhea.

Virulence gene profiles and molecular genetic characteristics of diarrheagenic Escherichia coli from a hospital in western China

BackgroundDiarrheagenic Escherichia coli (DEC) is one of the most important etiological agents of diarrheal diseases. In this study we investigated the prevalence, virulence gene profiles, antimicrobial resistance, and molecular genetic characteristics of DEC at a hospital in western China.MethodsA total of 110 Escherichia coli clinical isolates were collected from the First Affiliated Hospital of Chengdu Medical College from 2015 to 2016. Microbiological methods, PCR, antimicrobial susceptibility test, pulsed-field gel electrophoresis and multilocus sequence typing were used in this study.ResultsMolecular analysis of six DEC pathotype marker genes showed that 13 of the 110 E. coli isolates (11.82%) were DEC including nine (8.18%) diffusely adherent Escherichia coli (DAEC) and four (3.64%) enteroaggregative Escherichia coli (EAEC). The adherence genes fimC and fimH were present in all DAEC and EAEC isolates. All nine DAEC isolates harbored the virulence genes fyuA and irp2 and four (44.44%) also carried the hlyA and sat genes. The virulence genes fyuA, irp2, cnf1, hlyA, and sat were found in 100%, 100%, 75%, 50%, and 50% of EAEC isolates, respectively. In addition, all DEC isolates were multidrug resistant and had high frequencies of antimicrobial resistance. Molecular genetic characterization showed that the 13 DEC isolates were divided into 11 pulsed-field gel electrophoresis patterns and 10 sequence types.ConclusionsTo the best of our knowledge, this study provides the first report of DEC, including DAEC and EAEC, in western China. Our analyses identified the virulence genes present in E. coli from a hospital indicating their role in the isolated DEC strains’ pathogenesis. At the same time, the analyses revealed, the antimicrobial resistance pattern of the DEC isolates. Thus, DAEC and EAEC among the DEC strains should be considered a significant risk to humans in western China due to their evolved pathogenicity and antimicrobial resistance pattern.

Distribution of genes encoding virulence factors and multilocus variable-number tandem-repeat analysis (MLVA) of entero-aggregative Escherichia coli (EAEC) isolated in Iran from patients with diarrhoea.

Entero-aggregative Escherichia coli (EAEC) is one of the main causes of diarrhoea worldwide. Several virulence factors have been identified in EAEC. This study was conducted to investigate the distribution of virulence factor genes in EAEC strains isolated in Iran from children with diarrhoea, as well as the genetic similarity of these isolates. A total of 37 EAEC isolates were tested for the presence of 11 virulence genes by PCR, and the genetic relatedness of these strains was further determined by multilocus variable-number tandem-repeat analysis (MLVA). All EAEC isolates were typical EAEC. pic, set1A and set1B were the most prevalent genes, detected in 54.1 % of the isolates, followed by sat (43.2 %), astA (32.4 %), pet (24.3 %), agg4A (24.3 %), sepA (18.9 %), agg3A (13.5 %), sigA (8.1 %), aggA (8.1 %) and aafA (5.4 %). Using MLVA, the 37 isolates were divided into 32 types and classified into five clonal complexes. This study showed that EAEC is a heterogeneous group of E. coli possessing a broad range of virulence factors. There was no notable association between MLVA patterns and virulence profiles.

The Lectin Complement Pathway Is Involved in Protection Against Enteroaggregative Escherichia coli Infection

Enteroaggregative Escherichia coli (EAEC) causes acute and persistent diarrhea worldwide. Still, the involvement of host factors in EAEC infections is unresolved. Binding of recognition molecules from the lectin pathway of complement to EAEC strains have been observed, but the importance is not known. Our aim was to uncover the involvement of these molecules in innate complement dependent immune protection toward EAEC. Binding of mannose-binding lectin, ficolin-1, -2, and -3 to four prototypic EAEC strains, and ficolin-2 binding to 56 clinical EAEC isolates were screened by a consumption-based ELISA method. Flow cytometry was used to determine deposition of C4b, C3b, and the bactericidal C5b-9 membrane attack complex (MAC) on the bacteria in combination with different complement inhibitors. In addition, the direct serum bactericidal effect was assessed. Screening of the prototypic EAEC strains revealed that ficolin-2 was the major binder among the lectin pathway recognition molecules. However, among the clinical EAEC isolates only a restricted number (n = 5) of the isolates bound ficolin-2. Using the ficolin-2 binding isolate C322-17 as a model, we found that incubation with normal human serum led to deposition of C4b, C3b, and to MAC formation. No inhibition of complement deposition was observed when a C1q inhibitor was added, while partial inhibition was observed when ficolin-2 or factor D inhibitors were used separately. Combining the inhibitors against ficolin-2 and factor D led to virtually complete inhibition of complement deposition and protection against direct bacterial killing. These results demonstrate that ficolin-2 may play an important role in innate immune protection against EAEC when an appropriate ligand is exposed, but many EAEC strains evade lectin pathway recognition and may, therefore, circumvent this strategy of innate host immune protection.