ETEC Isolates Research Articles

Multidrug-resistant and clonal dispersion of enterotoxigenic Escherichia coli from ready-to-eat meat products in Duhok province, Iraq

This research evaluated the effluent proportion of E. coli and ETEC in RTE meat products, characterized the isolated strains' clonal relatedness, and determined their antibiotic resistance. 130 RTE products were gathered from various restaurants and street fast food vendors in Duhok and Zakho Province. The Isolates of E. coli identified by culture methods were confirmed as ETEC by multiplex PCR of the identified virulence genes. ERIC-PCR was applied to establish the clonal relationships between strains. The disk diffusion method performed the susceptibility of antibiotics on the isolated ETEC. Out of 130 examined samples, 39 (30%) isolates of E. coli and 16 (12.3%) ETEC were detected. Pan-fried burgers were revealed to be the most frequent contaminated sample type, with both E. coli and ETEC 50% and 23.3%, respectively (P≤0.05). A high clonal dispersion (12 genotypes) was observed among the isolated ETEC strains. A strong genetic linkage was discovered between a few isolates retrieved from the same sample type and within the strains from the same geographic source area. A high antibiotic resistance rate was observed with total resistance to Amoxicillin/clavulanate, Clarithromycin, Doxycycline, Erythromycin, and Clindamycin. Isolates from burger samples showed a higher resistance rate when compared with the other sample types (P≤0.05). Multi-drug resistance was noticed in all ETEC isolates. RTE meat products sold in our area have a high rate of clonally heterogeneous carrying multi-drug resistant ETEC and may constitute a significant public health risk.

Characterization of Selected Escherichia coli Pathovars and Their Antimicrobial Resistance Patterns among Diarrheal Children under the Age of Five Years from Machakos County, Kenya

Background: Diarrheal diseases constitute an important cause of death among children under the age of five years globally. These diseases are caused by diarrheagenic Escherichia coli including enteropathogenic Escherichia coli and enterotoxigenic Escherichia coli among other agents. Treatment and management of diarrheal diseases including EPEC and ETEC is complicated by rapidly developing problem of antimicrobial resistance. Methods: Stool samples were collected from children under the age of five years attending Machakos Level 5 hospital. Escherichia coli was isolated and identified by culture-based techniques followed by multiplex polymerase chain reaction using primers specific for virulence genes associated with EPEC and ETEC pathovars. Confirmed EPEC and ETEC pathovars were subjected to a panel of eight antimicrobial agents. Results: Both EPEC and ETEC were detected in 29/118 (24.6%) samples collected during the study period. Prevalence of EPEC was higher 18 (15.3%) compared to ETEC that was detected in 11 (9.3%) samples analyzed. ETEC appeared to be more resistant to ampicillin (90.9%, 66.7%), trimethoprim (81.8%, 77.8%), gentamicin (45.5%, 22.2%), chloramphenicol (27.3%, 16.3%), cefuroxime (18.2%, 5.6%) and ciprofloxacin (9.1%, 5.6%) compared to EPEC respectively. On the other hand, EPEC displayed higher resistance against nalidixic acid (38.9%, 36.4%) and tetracycline (33.3%, 18.2%) compared to ETEC isolates. Conclusion: The role of EPEC and ETEC as a cause of infantile diarrhea cannot be underestimated in Machakos County, Kenya since they are both pathogenic and resistant to commonly used antimicrobial agents.

Co-infection With Chromosomally-Located bla CTX-M-14 and Plasmid-Encoding bla CTX-M-15 in Pathogenic Escherichia coli in the Republic of Korea.

The emergence of third-generation cephalosporin resistance in Escherichia coli is increasing at an alarming rate in many countries. Thus, the aim of this study was to analyze co-infecting blaCTX-M-producing pathogenic E. coli isolates linked to three school outbreaks. Among 66 E. coli isolates, 44 were identified as ETEC O25, an ETEC isolate serotype was O2, and the other 21 were confirmed as EAEC O44. Interestingly, six patients were co-infected with EAEC O44 and ETEC O25. For these isolates, molecular analysis [antibiotic susceptibility testing, identification of the β-lactamase gene, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE)] was performed for further characterization. In addition, the transmission capacity of blaCTX-M genes was examined by conjugation experiments. Whole-genome sequencing (WGS) was performed on representative EAEC O44 and ETEC O25 isolates associated with co-infection and single-infection. All isolates were resistant to cefotaxime and ceftriaxone. All EAEC isolates carried the blaCTX-M-14 gene and all ETEC isolates the blaCTX-M-15 gene, as detected by multiplex PCR and sequencing analysis. Sequence type and PFGE results indicated three different patterns depending on the O serotype. WGS results of representative isolates revealed that the ETEC O25 strains harbored blaCTX-M-15 located on IncK plasmids associated with the ΔblaTEM-blaCTX-M-15-orf477 transposon. The representative EAEC O44 isolates carried blaCTX-M-14 on the chromosome, which was surrounded by the ISEcp1-blaCTX-M-14-IS903 transposon. To the best of our knowledge, this is the first report of co-infection with chromosomally located blaCTX-M-14 and plasmid-encoding blaCTX-M-15 in pathogenic E. coli. Our findings indicate that resistance genes in clinical isolates can spread through concurrent combinations of chromosomes and plasmids.

Conservation and global distribution of non-canonical antigens in Enterotoxigenic Escherichia coli.

BackgroundEnterotoxigenic Escherichia coli (ETEC) cause significant diarrheal morbidity and mortality in children of resource-limited regions, warranting development of effective vaccine strategies. Genetic diversity of the ETEC pathovar has impeded development of broadly protective vaccines centered on the classical canonical antigens, the colonization factors and heat-labile toxin. Two non-canonical ETEC antigens, the EtpA adhesin, and the EatA mucinase are immunogenic in humans and protective in animal models. To foster rational vaccine design that complements existing strategies, we examined the distribution and molecular conservation of these antigens in a diverse population of ETEC isolates.MethodsGeographically diverse ETEC isolates (n = 1159) were interrogated by PCR, immunoblotting, and/or whole genome sequencing (n = 46) to examine antigen conservation. The most divergent proteins were purified and their core functions assessed in vitro.ResultsEatA and EtpA or their coding sequences were present in 57.0% and 51.5% of the ETEC isolates overall, respectively; and were globally dispersed without significant regional differences in antigen distribution. These antigens also exhibited >93% amino acid sequence identity with even the most divergent proteins retaining the core adhesin and mucinase activity assigned to the prototype molecules.ConclusionsEtpA and EatA are well-conserved molecules in the ETEC pathovar, suggesting that they serve important roles in virulence and that they could be exploited for rational vaccine design.

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.

Antibiotic resistance, ESBL genes, integrons, phylogenetic groups and MLVA profiles of Escherichia coli pathotypes isolated from patients with diarrhea and farm animals in south-east of Iran.

The aims of this study were to investigate the prevalence, antibiotic resistance, presence of class 1 and 2 integrons, Extended Spectrum β-Lactamases (ESBL) genes, phylogenetic group and epidemiological relationships of EPEC, ETEC and EHEC pathotypes isolated from patients with diarrhea and farm animals in south east region of Iran. A total of 671 diarrheagenic E. coli (DEC) were collected from stool samples of 395 patients with diarrhea and 276 farm cattles and goats. Presence of EPEC, ETEC and EHEC were identified using multiplex-PCR employing primers targeted the shiga toxin (stx), intimin (eae), bundle forming pili (bfp), and enterotoxins (lt and st) genes. The highest proportion of the patients (64%) were children under age 1-15 year (p ≤ 0.05). Among the isolates, atypical EPEC was detected in 26 patients and 14 animal stool samples, while typical EPEC was found in 2 cattles. ETEC isolates were detected in stools of 13 patients and 4 EHEC was identified in 3 goats and one cattle. The isolates were checked for susceptibility to 14 antibiotics. 50% (n = 13) of EPEC and 61.5% (n =8) of ETEC showed multi-drug resistance (MDR) profiles and one EPEC was found to be extensive drug resistant (XDR). In contrast, EHEC isolates were susceptible to the majority of antimicrobial agents. The MDR isolates were positive for blaTEM and blaCTX-M ESBL genes and carried class 1 integrons. Further study on the biofilm formation indicated that, 3 out of 4 EHEC isolates showed strong biofilm, while other pathotypes had either moderate, weak or no biofilm activity. Majority of EPEC isolates were belonged to phylogenetic group B1, all except one ETEC were classified as phylogenetic group A and two EHEC were belonged to phylogroup D, respectively. A multilocus variable tandem repeat analysis (MLVA) exhibited 22 distinct patterns. In conclusion, MLVA data showed high clonal diversity. Presence of EHEC in animal origins pose public health concern in this region.

Study on the presence of resistant diarrheagenic pathotypes in Escherichia coli isolated from patients with urinary tract infection.

This article aimed to analyze the diarrheagenic potential of E. coli isolated from urinary tract infection (UTI) and to recognize the presence of antibiotic resistance genes. The marked genome plasticity of Escherichia coli has allowed the emergence of resistant pathogenic strains displaying an unusual arrangement of genes. In this cross-sectional study, 110 E. coli were isolated from patients with the symptoms of UTI in Sanandaj, west of Iran between July and September - 2015. The isolates were examined by the disk diffusion method for antibiotic susceptibility test and by polymerase chain reaction for the presence of genes characteristic of diarrheagenic E. coli (DEC), Uropathogenic E. coli (UPEC) virulence genes, extended-spectrum β-lactamase bla CTX-M and plasmid-mediated quinolone resistance determinants, qnrA, qnrB, and qnrS. The most and the least effective antibiotics were nitrofurantoin and cefotaxime (96.4% and 27.3% sensitivity, respectively). Of the 110 UTI isolates, 57.3% carried diarrheagenic genes. The bundle-forming pilus bfpA was the most prevalent diarrheagenic gene (39.1%). The most commonly detected DEC pathotype was enterotoxigenic E. coli (-ETEC, 12.7%). All the pathotypes carried the bla CTX-M and qnr. The -UPEC hly hemolysin and pap adhesin genes were mainly detected among ETEC isolates. Our results indicated the presence of resistant diarrheagenic pathotypes in UTI-associated E. coli. Such isolates may have the capacity of causing both extraintestinal and intestinal infections. Based on our knowledge, this is the first report of the presence of qnr in ETEC from urine.

Virulence genes and antibiotic resistance profile of shiga toxin producing Escherichia coli and enterotoxigenic Escherichia coli from diarrhoeic cattles and human handlers in Asom (India)

The present study was undertaken to isolate and characterize STEC and ETEC from diarrhoeic calves and humans in Asom, India. Out of the 134 presumptive Escherichia coli isolates picked from 80 diarrhoeic calves, 31 isolates could be serotyped into 23 different serogroups; and out of 19 strains of E. coli from human beings, 13 strains could be serotyped into 10 different serogroups. O157 from cattle and O131 from humans were the frequently encountered serogroups. The virulence gene profile of the STEC and ETEC isolates showed that the prevalence of stx2 gene and the emergence of multi drug resistant STEC and ETEC isolates in high proportion indicates that they may act as an important reservoir posing a possible threat to public health and may complicate future therapeutic options.

Studies on the serum types and identification efficiency on Diarrheagenic Escherichia coli isolated from diarrhea patients, in Zhejiang province

Objective: To investigate the serotypes of Diarrheagenic Escherichia coli (DEC) isolated from diarrheal patients in Zhejiang province and to explore the identification efficiency of serological screening methods. Methods: Serological agglutination tests were carried out in 696 strains of DEC (through the identification of virulence genes) which were selected from the Infectious Diarrhea Pathogen Monitoring Network Strain Bank of Zhejiang province, from July 2009 to June 2013. Results of virulence genes, serological identification and classification were compared. Results: Among the 696 isolates of DEC, O antigen type was identified in 288 (41.4%) isolates which belonging to 35 different 'O' serum types. H antigen was seen in 171 (24.6%) isolates and determined as having 21 types. The agglutination rates of EAEC, ETEC, EPEC and EHEC isolates were 31.9% (130/408), 70.6% (127/180), 31.5% (29/92) and 14.3% (2/14), respectively and belonged to 30, 18, 15 kinds of 'O' sero-groups, respectively. One EHEC isolate was identified as O157∶H7. Serum groups were diverse for EAEC and EPEC, while relatively concentrated on ETEC. Different types of DEC might belong to the same sero-group or type. Among the 74 strains of DEC available for classification serologically, 41 isolates were in consistent with virulence gene identification and another 33 strains were not. Conclusions: The sero-group/type of DEC strains in Zhejiang were varied. Based on the serological screening method alone, DEC classification might end in getting the wrong answer, thus we would recommend the use of virulence gene for the purpose of identification.

Development of lipopolysaccharide-mimicking peptides and their immunoprotectivity against Vibrio cholerae serogroup O1.

Vibrio cholerae serogroup O1 is the main causative agent of cholera diseases defined by life threatening rice watery diarrhea. Cholera routine vaccination has failed in controlling epidemics in developing countries because of their hard and expensive production. In this study, our aim was to investigate phage displayed mimotopes that could mimic V. cholerae lipopolysaccharide (LPS). Although LPS of Vibrio, as an endotoxin, can stimulate the immune system, thereby making it a suitable candidate for cholera vaccine, its toxicity remains as a main problem. Phage particles displaying 12 amino acid peptides were selected from phage library mimicking the antigenic epitopes of LPS from vibrio. The screening was carried out using single-domain antibody fragment VHH against LPS as target through three rounds of selection. Three clones with highest affinity to VHH were selected. To find out a new and efficient vaccine against cholera, these three phage particles containing high-affinity peptides were administered to mice to investigate the active and passive immunity. Out of 20 particles, three showed the highest affinity toward VHH. ELISA was carried out with immunized mice sera using LPS and three selected phages particles individually. ETEC, Shigella sonnei, and clinical isolates were used as bacterial targets. These three selected phages (individually or in combination) could stimulate mice immune system producing active and passive immunity. The mice immunized with phage particles could protect about 14 LD50 of V. cholerae. In conclusion, these peptides are mimicking LPS and can potentially act as vaccine candidates against V. cholerae. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Vaccinating with conserved Escherichia coli antigens does not alter the mouse intestinal microbiome.

BackgroundEnterotoxigenic Escherichia coli (ETEC) causes diarrheal disease. Antigenic and structural heterogeneity among ETEC colonization factors has complicated vaccine development efforts. Identifying and characterizing conserved ETEC antigens that induce protective immunity is therefore of interest. We previously characterized three proteins (MipA, Skp, and ETEC_2479) that protected mice in an intranasal ETEC challenge model after vaccination. However, these proteins are conserved not only in multiple ETEC isolates, but also in commensal bacteria. While the impact of inactivated viral vaccines and live-attenuated bacterial vaccines on the host microbiota have been examined, the potential impact of using subunit vaccines consisting of antigens that are also encoded by commensal organisms has not been investigated.FindingsWe addressed this issue by characterizing changes to mouse intestinal microbiomes as a function of vaccination. We failed to observe significant changes to mouse health, to mouse weight gain as a function of time, or to the diversity or richness of mouse intestinal microbiomes, as measured by analyzing alpha- and beta-diversity, as well as overall community structure, before and after vaccination.ConclusionsWe conclude that despite the conservation of MipA, Skp, and ETEC_2479 among Gram-negative bacteria, vaccination with these antigens fails to alter significantly the host intestinal microbiome.

Impact of urban contamination of the La Paz River basin on thermotolerant coliform density and occurrence of multiple antibiotic resistant enteric pathogens in river water, irrigated soil and fresh vegetables.

La Paz River in Andean highlands is heavily polluted with urban run-off and further contaminates agricultural lowlands and downstream waters at the Amazon watershed. Agricultural produce at this region is the main source of vegetables for the major Andean cities of La Paz and El Alto. We conducted a 1 year study, to evaluate microbial quality parameters and occurrence of multiple enteropathogenic bacteria (Enterohemorrhagic E. coli—EHEC, Enteroinvasive E. coli or Shigella—EIEC/Shigella, Enteroaggregative E. coli—EAEC, Enteropathogenic E. coli—EPEC Enterotoxigenic E. coli—ETEC and Salmonella) and its resistance to 11 antibiotics. Four sampling locations were selected: a fresh mountain water reservoir (un-impacted, site 1) and downstream sites receiving wastewater discharges (impacted, sites 2–4). River water (sites 1–4, N = 48), and soil and vegetable samples (site 3, N = 24) were collected during dry (April–September) and rainy seasons (October–March). Throughout the study, thermotolerant coliform density values at impacted sites greatly exceeded the guidelines for recreational and agricultural water uses. Seasonal differences were found for thermotolerant coliform density during dry season in water samples nearby a populated and hospital compound area. In contrast to the un-impacted site, where none of the tested enteropathogens were found, 100 % of surface water, 83 % of soil and 67 % of vegetable samples at impacted sites, were contaminated with at least one enteropathogen, being ETEC and Salmonella the most frequently found. ETEC isolates displayed different patterns of toxin genes among sites. The occurrence of enteropathogens was associated with the thermotolerant coliform density. At impacted sites, multiple enteropathogens were frequently found during rainy season. Among isolated enteropathogens, 50 % were resistant to at least two antibiotics, with resistance to ampicillin, nalidixic acid, trimethoprim–sulfamethoxazole and tetracycline commonly present. Moreover, some Salmonella isolates were distinguished by their multi-resistance to ≥8 antibiotics, within soil and vegetable samples. Overall, this study demonstrates that La Paz River—an affluent of the Amazon macrobasin—is heavily polluted along the year with a high density of thermotolerant coliforms and is a reservoir of multiple antibiotic resistant enteropathogens, present in river water, soil and vegetables. These data highlight health risk associated with food and waterborne diseases at the region.

Examination of the Enterotoxigenic Escherichia coli Population Structure during Human Infection.

ABSTRACTEnterotoxigenic E. coli (ETEC) can cause severe diarrhea and death in children in developing countries; however, bacterial diversity in natural infection is uncharacterized. In this study, we explored the natural population variation of ETEC from individuals with cholera-like diarrhea. Genomic sequencing and comparative analysis of multiple ETEC isolates from twelve cases of severe diarrhea demonstrated clonal populations in the majority of subjects (10/12). In contrast, a minority of individuals (2/12) yielded phylogenomically divergent ETEC isolates. Detailed examination revealed that isolates also differed in virulence factor content. These genomic data suggest that severe, cholera-like ETEC infections are largely caused by a clonal population of organisms within individual patients. Additionally, the isolation of similar clones from geographically and temporally dispersed cases with similar clinical presentations suggests that some isolates are particularly suited for virulence. The identification of multiple genomically diverse isolates with variable virulence factor profiles from a single subject highlights the dynamic nature of ETEC, as well as a potential weakness in the examination of cultures obtained from a single colony in clinical settings. These findings have implications for vaccine design and provide a framework for the study of population variation in other human pathogens.

Draft Whole-Genome Sequences of 10 Serogroup O6 Enterotoxigenic Escherichia coli Strains.

Entertotoxigenic Escherichia coli (ETEC) is a major cause of global diarrhea, resulting in approximately 200 million occurrences and 300,000 to 400,000 deaths annually, primarily in children under the age of five. Here, we announce the release of the draft genomes of 10 ETEC isolates belonging to serogroup O6.

Coverage of diarrhoea‐associated Escherichia coli isolates from different origins with two types of phage cocktails

Eighty-nine T4-like phages from our phage collection were tested against four collections of childhood diarrhoea-associated Escherichia coli isolates representing different geographical origins (Mexico versus Bangladesh), serotypes (69 O, 27 H serotypes), pathotypes (ETEC, EPEC, EIEC, EAEC, VTEC, Shigella), epidemiological settings (community and hospitalized diarrhoea) and years of isolation. With a cocktail consisting of 3 to 14 T4-like phages, we achieved 54% to 69% coverage against predominantly EPEC isolates from Mexico, 30% to 53% against mostly ETEC isolates from a prospective survey in Bangladesh, 24% to 61% against a mixture of pathotypes isolated from hospitalized children in Bangladesh, and 60% coverage against Shigella isolates. In comparison a commercial Russian phage cocktail containing a complex mixture of many different genera of coliphages showed 19%, 33%, 50% and 90% coverage, respectively, against the four above-mentioned collections. Few O serotype-specific phages and no broad-host range phages were detected in our T4-like phage collection. Interference phenomena between the phage isolates were observed when constituting larger phage cocktails. Since the coverage of a given T4-like phage cocktail differed with geographical area and epidemiological setting, a phage composition adapted to a local situation is needed for phage therapy approaches against E. coli pathogens.

Pathogenicity and Phenotypic Characterization of Enterotoxigenic Escherichia coli Isolates from a Birth Cohort of Children in Rural Egypt

Enterotoxigenic Escherichia coli (ETEC) has consistently been the predominant bacterial cause of diarrhea in many birth cohort- and hospital-based studies conducted in Egypt. We evaluated the pathogenicity of ETEC isolates in a birth cohort of children living in a rural community in Egypt. Between 2004 and 2007, we enrolled and followed 348 children starting at birth until their second year of life. A stool sample and two rectal swabs were collected from children during twice-weekly visits when they presented with diarrhea and were collected every 2 weeks if no diarrhea was reported. From routine stool cultures, five E. coli-like colonies were screened for ETEC enterotoxins using a GM1 enzyme-linked immunosorbent assay (ELISA). The isolates were screened against a panel of 12 colonization factor antigens (CFAs) by a dot blot assay. A nested case-control study evaluated the association between initial or repeat excretion of ETEC and the occurrences of diarrhea. The pathogenicity of ETEC was estimated in symptomatic children compared to that in asymptomatic controls. ETEC was significantly associated with diarrhea (crude odds ratio, 1.37; 95% confidence interval [CI], 1.24 to 1.52). The distribution of ETEC enterotoxins varied between the symptomatic children (44.2% heat-labile toxin [LT], 38.5% heat-stable toxin [ST], and 17.3% LT/ST) and asymptomatic children (55.5% LT, 34.6% ST, and 9.9% LT/ST) (P < 0.001). The CFAs CFA/I (n = 61), CS3 (n = 8), CS1 plus CS3 (n = 24), CS2 plus CS3 (n = 18), CS6 (n = 45), CS5 plus CS6 (n = 11), CS7 (n = 25), and CS14 (n = 32) were frequently detected in symptomatic children, while CS6 (n = 66), CS12 (n = 51), CFA/I (n = 43), and CS14 (n = 20) were detected at higher frequencies among asymptomatic children. While all toxin phenotypes were associated with diarrheal disease after the initial exposure, only ST and LT/ST-expressing ETEC isolates (P < 0.0001) were associated with disease in repeat infections. The role of enterotoxins and pathogenicity during repeat ETEC infections appears to be variable and dependent on the coexpression of specific CFAs.

Potential antibacterial activity of berberine against multi drug resistant enterovirulent Escherichia coli isolated from yaks (Poephagus grunniens) with haemorrhagic diarrhoea

ObjectiveTo evaluate the antimicrobial efficacy of berberine, a plant alkaloid. MethodsFive multi-drug resistant (MDR) STEC/EPEC and five MDR ETEC isolates from yaks with haemorrhagic diarrhoea were selected for the study. Antibacterial activity of berberine was evaluated by broth dilution and disc diffusion methods. The binding kinetics of berberine to DNA and protein was also enumerated. ResultsFor both categories of enterovirulent Escherichia coli (E. coli) isolates, berberine displayed the antibacterial effect in a dose dependent manner. The MIC50 of berberine chloride for STEC/EPEC isolates varied from 2.07 μM to 3.6 μM with a mean of (2.95 ± 0.33) μM where as for ETEC strains it varied from 1.75 to 1.96 μM with a mean of (1.87 ± 0.03) μM. Berberine bind more tightly with double helix DNA with Bmax and Kd of (24.68±2.62) and (357.8±57.8), respectively. Berberine reacted with protein in comparatively loose manner with Bmax and Kd of (18.9±3.83) and (286.2±113.6), respectively. ConclusionsThe results indicate clearly that berberine may serve as a good antibacterial against multi drug resistant E. coli.

Molecular Characterization of Enterotoxigenic Escherichia coli Isolates Recovered from Children with Diarrhea during a 4-Year Period (2007 to 2010) in Bolivia

Enterotoxigenic Escherichia coli (ETEC) is an important cause of childhood diarrhea. This study aimed to characterize ETEC strains isolated from Bolivian children aged <5 years according to enterotoxin profile, colonization factors (CFs), suggested virulence genes, and severity of disease. A total of 299 ETEC isolates recovered from children with diarrhea and 55 ETEC isolates from children without diarrhea (controls) were isolated over a period of 4 years. Strains expressing heat-labile toxin (LT) or heat-stable toxin (ST) alone were about equally common and twice as common as ETEC producing both toxins (20%). ETEC strains expressing human ST (STh) were more common in children aged <2 years, while ETEC strains expressing LT plus STh (LT/STh) were more frequent in 2- to 5-year-old children. Severity of disease was not related to the toxin profile of the strains. CF-positive isolates were more frequently identified in diarrheal samples than in control samples (P = 0.02). The most common CFs were CFA/I and CS14. CFA/I ETEC strains were more frequent in children aged <2 years than CS1+CS3 isolates and CS14 isolates, which were more prevalent in 2- to 5-year-old children. The presence of suggested ETEC virulence genes (clyA, eatA, tia, tibC, leoA, and east-1) was not associated with disease. However, east-1 was associated with LT/STh strains (P < 0.001), eatA with STh strains (P < 0.001), and tia with LT/STh strains (P < 0.001). A minor seasonal peak of ETEC infections was identified in May during the cold-dry season and coincided with the peak of rotavirus infections; this pattern is unusual for ETEC and may be important for vaccination strategies in Bolivia.

Real-Time PCR-Based Mismatch Amplification Mutation Assay for Specific Detection of CS6-Expressing Allelic Variants of Enterotoxigenic Escherichia coli and Its Application in Assessing Diarrheal Cases and Asymptomatic Controls

Enterotoxigenic Escherichia coli (ETEC) expressing the colonization factor CS6 is widespread in many developing countries, including India. The different allelic variants of CS6, caused by point mutations in its structural genes, cssA and cssB, are designated AIBI, AIIBII, AIIIBI, AIBII, and AIIIBII. A simple, reliable, and specific mismatch amplification mutation assay based on real-time quantitative PCR (MAMA-qPCR) was developed for the first time for the detection of CS6-expressing ETEC, along with the identification of allelic variations. The assay was based on mismatched nucleotide incorporation at the penultimate base at the 3' ends of the reverse primers specific for cssA and cssB and was validated using 38 CS6-expressing ETEC isolates. This strategy was effective in detecting all the alleles containing single-nucleotide polymorphisms. Using MAMA-qPCR, we also tested CS6 allelic variants in 145 ETEC isolates from children with acute diarrhea and asymptomatic infections, with the latter serving as controls. We observed that the AIBI and AIIIBI allelic variants were mostly associated with cases rather than controls, whereas the AIIBII variants were detected mostly in controls. In addition, the AIBI and AIIIBI alleles were frequently associated with ETEC harboring the heat-stable toxin gene (est) alone or with the heat-labile toxin gene (elt), whereas the AIIBII allele was predominant in ETEC isolates harboring the elt gene. This study may help in understanding the association of allelic variants in CS6-expressing ETEC with the clinical features of diarrhea, as well as in ETEC vaccine studies.

Detection of virulence factors of Escherichia coli focused on prevalence of EAST1 toxin in stool of diarrheic and non-diarrheic piglets and presence of adhesion involving virulence factors in astA positive strains

Between 2005 and 2009, a total of 800 Escherichia coli strains isolated from piglets with diarrhea were tested for the presence of enteroaggregative heat-stable enterotoxin EAST1, heat-labile (LT) and heat-stable enterotoxins (STa) and shigatoxin (Stx2e) by PCR with the purpose of investigating the present distribution of virulence factors on swine farms in the Czech Republic. The isolates were analyzed for their O-serogroup, fimbrial (K88, K99, 987P, F41, F18) and nonfimbrial adhesins (adhesin involved in diffuse adherence AIDA and porcine attaching and effacing-associated factor PAA). The detection rates of ETEC and STEC isolates were 36.5% and 7.75%, respectively, which implies that ETEC play the major role in E. coli infections in Czech herds. Generally, the most common serotype was O149:K88 which possessed genetic determinants for LT and EAST1. None of the tested E. coli isolates was positive for genes K99, 987P and F41. It was shown that out of 800 E. coli strains isolated from pigs, 277 were EAST1 positive and 74% from the latter were identified as ETEC. Of the fimbrial adhesins, K88 and F18 were commonly detected. Over 80% of K88/EAST1 positive strains possessed the gene for paa. We detected no EAE isolate positive for fimbrial adhesins or PAA and AIDA. The AIDA was more often associated with F18 than with K88. The gene astA was also identified among E. coli isolates of non-diarrheic piglets. We tested rectal swab samples collected from apparently healthy piglets on three farms. On all farms, E. coli astA positive strains (26.66%, 90.00% and 46.66% astA positive animals) were isolated. Our results showed a significantly higher prevalence of astA positive E. coli isolates among apparently healthy piglets in comparison with diarrheic piglets. The question remains as to what is the role of the astA gene in the pathogenesis of porcine colibacillosis and as a virulence factor.