Real-Time Multiplex Polymerase Chain Reaction with High-Resolution Melting-Curve Analysis for the Diagnosis of Enteric Infections Associated with Diarrheagenic Escherichia coli

Introduction: Although diarrheagenic Escherichia coli (DEC) strains are important bacterial causative agents of diarrhoea, they are not routinely sought as stool pathogens in clinical laboratories as conventional microbiological testing are unable to distinguish between normal flora and pathogenic strains of E. coli. This study was undertaken to determine the prevalence of DEC pathotypes amongst children with and without diarrhoea and to detect specific virulent genes present in different DEC pathotypes, using real-time multiplex polymerase chain reaction (PCR) with high-resolution melting (HRM) technology. Materials and Methods: Stool samples were obtained from cases and controls. Using a set of conventional biochemical tests, E. coli strains were identified. Further, these isolates were subjected to multiplex PCR system for the detection of virulence genes of different pathotypes of DEC. Real-time multiplex PCR was performed for the detection of specific virulent genes of DEC pathotypes, using Rotor-Gene Q instrument (Qiagen) having High-resolution Melt analyser using Type-it HRM PCR kit (Qiagen) containing EvaGreen fluorescent intercalating dye. Results: In this study, we had successfully standardised two multiplex PCR assays which were found to be effective for direct detection of enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), enterotoxigenic E. coli (ETEC) and enteroinvasive E. coli (EIEC). A total of 42 DEC strains were detected at an overall rate of 19.3% (n = 42), from the total 217 E. coli isolates recovered from the cases (n = 39, 17.9%) and control (n = 3, 3.8%) groups. Amongst the 42 DEC pathotypes (39 from cases and 3 from controls), EPEC (10%), EAEC (8.82%), ETEC (2.94%) and EIEC (1.18%) were found in children with diarrhoea (cases) and in children without diarrhoea (control) only EAEC (2.13%) and EPEC (4.26%) were detected. Age distribution, gender variation, seasonal variation and clinical features were also analysed Conclusion: This study helped evaluate the prevalence of DEC amongst children (<18 years of age) with and without diarrhoea using multiplex real-time PCR with HRM analysis.     

Diarrheagenic Escherichia coli pathotypes investigation revealed atypical enteropathogenic E. coli as putative emerging diarrheal agents in children living in Botucatu,São Paulo State,Brazil

The aim of the present study was to investigate the prevalence of Diarrheagenic Escherichia coli (DEC) pathotypes, a leading cause of diarrhea worldwide, among diarrheal and healthy children, up to 5 years of age, living in the city of Botucatu, São Paulo, Brazil. DEC, investigated by PCR detection of virulence factor‐encoding genes associated with the distinct pathotypes, was isolated from 18.0% of the patients, and 19.0% of the controls, with enteroaggregative E. coli (EAEC), the most frequent pathotype, being detected in equal proportion between patients and controls (10.0%). Among the enteropathogenic E. coli (EPEC) isolates, only one isolate was able to produce the localized adherence pattern to HeLa cells, being thus the only typical EPEC identified. All the remaining EPEC were classified as atypical (aEPEC), and detected in 8.0% and 8.5% of the patients and controls, respectively. Regarding the serotypes, 26.5% of the analyzed EPEC isolates belonged to classical EPEC‐serogroups, and the only two STEC found were serotyped as O26:H11 (patient) and O119:H7 (control). Antimicrobial susceptibility tests revealed that 43.6%, 29.5% and 2.6% of the DEC isolates were resistant to ampicillin, cotrimoxazole and gentamicin, respectively. Our data indicate that EAEC remains prevalent among children living in Botucatu, and revealed atypical EPEC as emerging putative diarrheal agents in this geographical region.     

Flooding adds pathogenic Escherichia coli strains to the water sources in southern Khyber Pakhtunkhwa,Pakistan

Purpose: Seasonal rains in Pakistan result in heavy floods across the country, whereby faecal contaminants will be added to the water bodies and cause numerous food-borne outbreaks. The present study was aimed to determine the prevalence of diarrheagenic Escherichia coli (DEC) strains in the water sources. Materials and Methods: Two hundred water samples collected during (2011–2012) were processed for the isolation of E. coli (EC) strains. EC strains were further analysed for antibiotic susceptibility patterns, and pathogroups-specific virulence factors stx1, stx2, stx2c, eae, tir, hlyA, bfpA, estA and eltA were detected using multiplex polymerase chain reaction. Results: Thirty-three percent of the water samples were contaminated with EC pathotypes. Fifty percent (33/66) of the DEC pathotypes were identified as enterotoxigenic EC (ETEC). Seventy-two percent (13/18) of the enteropathogenic EC (EPEC) strains were identified as typical EPEC and 28% (5/18) as atypical EPEC. Eleven percent (7/66) of the Shiga toxin EC (STEC) isolates carried a combination of stx1 and stx2 genes. Summer was found as a peak season with 47% (31/66) for EC pathogroups’ activities. Eighty-nine percent of the strains showed resistance against tetracycline. Conclusion: ETEC and EPEC are the primary causes of water contamination in southern regions of Khyber Pakhtunkhwa province, Pakistan. Firm adherence to the prescribed drugs can decrease trends in antibiotic resistance.     

Serotypes,genotypes and antimicrobial resistance patterns of human diarrhoeagenic Escherichia coli isolates circulating in southeastern China

Diarrhoeagenic Escherichia coli (DEC) infection is a major health problem in developing countries. The prevalence and characteristics of DEC have not been thoroughly investigated in China. Consecutive faecal specimens from outpatients with acute diarrhoea in nine sentinel hospitals in southeastern China were collected from July 2009 to June 2011. Bacterial and viral pathogens were detected by culture and RT-PCR, respectively. DEC isolates were further classified into five pathotypes using multiplex PCR. The O/H serotypes, sequence types (STs) and antimicrobial susceptibility profiles of the DEC isolates were determined. A total of 2466 faecal specimens were collected, from which 347 (14.1%) DEC isolates were isolated. DEC was the dominant bacterial pathogen detected. The DEC isolates included 217 EAEC, 62 ETEC, 52 EPEC, 14 STEC, one EIEC and one EAEC/ETEC. O45 (6.6%) was the predominant serotype. Genotypic analysis revealed that the major genotype was ST complex 10 (87, 25.6%). Isolates belonging to the serogroups or genotypes of O6, O25, O159, ST48, ST218, ST94 and ST1491 were highly susceptible to the majority of antimicrobials. In contrast, isolates belonging to O45, O15, O1, O169, ST38, ST226, ST69, ST31, ST93, ST394 and ST648 were highly resistant to the majority of antimicrobials. DEC accounted for the majority of bacterial pathogens causing acute diarrhoea in southeastern China, and it is therefore necessary to test for all DEC, not only the EHEC O157:H7. Some serogroups or genotypes of DEC were highly resistant to the majority of antimicrobials. DEC surveillance should be emphasized.     

Interleukin-8 Response in an Intestinal HCT-8 Cell Line Infected with Enteroaggregative and Enterotoxigenic Escherichia coli

This study examined the interleukin-8 (IL-8) response of the intestinal adenocarcinoma HCT-8 cell line to infection with enteroaggregative and enterotoxigenic Escherichia coli pathotypes isolated from patients with travelers' diarrhea. Individual diarrheagenic E. coli strains (enteroaggregative E. coli [EAEC]; n = 30), heat-stable enterotoxin (ST)-producing enterotoxigenic E. coli (ETEC ST; n = 11), heat-labile enterotoxin (LT)-producing enterotoxigenic E. coli (ETEC LT; n = 10), and ST- and LT-producing enterotoxigenic E. coli (ETEC ST:LT; n = 8) were coincubated with HCT-8 cells for 3 h. Tissue culture supernatants were assayed for IL-8 content by enzyme-linked immunosorbent assay. Fifty percent of EAEC (72% of those EAEC carrying the virulence factors aggR, aggA, and aspU and 40% of those EAEC not carrying virulence factors) and 64% of ETEC ST elicited IL-8 production. In contrast, 10% of ETEC LT elicited the production of IL-8 above baseline. These results suggest that (i) the HCT-8 cell line infection model can be used as a tool to differentiate proinflammatory E. coli from noninflammatory isolates; (ii) EAEC has a heterogeneous ability to induce the production of IL-8, and this may be associated with the presence of virulence factors; and (iii) ETEC ST can elicit an inflammatory response and helps explain our earlier findings of increased fecal IL-8 in patients with ETEC diarrhea.     

Detection of diarrheagenic Escherichia coli by multiplex PCR

Background: Diarrheagenic E.coli (DEC) are an important cause of childhood diarrhea.Identification of DEC strains needs to detect factors that determine the virulence of these organisms. There is not much data regarding the importance of DEC as a cause of diarrhea in children in India.The prevalence of DEC in children belowfive years with and without diarrhea was studied using two multiplex PCR assays. Materials and Methods: Two multiplex polymerase chain reaction assays were used to detect genes of five types of DEC.The targets selected for each category were eae and bfpA (bundle-forming pilus) forEnteropathogenic E.coli (EPEC), hlyA for Enterohemorrhagic E.coli (EHEC), elt and stla for Enterotoxigenic E.coli (ETEC), CVD432 for Enteroaggregative E.coli (EAEC) and ial for Enteroinvasive E.coli (EIEC). Results: In 200 children with diarrhea 52 (26%) DEC infections were found. Among 100 controls 8 (8%) DEC infections were found. EAEC was the most common DEC by multiplex PCR both in cases (26, 13%)and controls (5,5%), followed byEPEC seen in 16% cases and 3% controls. ETEC and EIEC were found in 7 (3.5%) and 3 (1.5%) of the diarrheal cases. EIEC and ETEC were not detected in the control cases. EHEC was not isolated from either the diarrheal or control cases. Conclusion: DEC strains are a significant cause of diarrhea in children. The two Multiplex PCR assays can be used for the detection of DEC in routine diagnostic laboratories. These assays are specific and sensitive for the rapid detection of DEC. EAEC was the most frequent pathotype in the population under study.     

A prospective study of travellers' diarrhoea: analysis of pathogen findings by destination in various (sub)tropical regions

ObjectivesEighty million travellers visiting (sub)tropical regions contract travellers' diarrhoea (TD) each year, yet prospective data comparing the prevalence of TD pathogens in various geographical regions are scarce. Our recent study using modern molecular methods found enteropathogenic (EPEC) and enteroaggregative (EAEC) Escherichia coli to be the most frequent pathogens, followed by enterotoxigenic E. coli (ETEC) and Campylobacter. We revisited our data to compare the findings by geographical region.MethodsA total of 459 prospectively recruited travellers provided stool samples and completed questionnaires before and after visiting destinations in various geographical regions. A multiplex quantitative real-time PCR assay was used to analyse Salmonella, Yersinia, Campylobacter jejuni/Campylobacter coli, Shigella, Vibrio cholerae, EPEC, EAEC, ETEC, enterohaemorrhagic E. coli and enteroinvasive E. coli.ResultsTD was contracted by 69% (316/459) of the subjects; EPEC and EAEC outnumbered ETEC and Campylobacter in all regions. Multiple pathogens were detected in 42% (133/316) of the samples. The proportions of all pathogens varied by region. The greatest differences were seen for Campylobacter: while relatively frequent in South Asia (n = 11; 20% of the 55 with TD during travel) and Southeast Asia (15/84, 15%), it was less common in East and West Africa (5/71, 7% and 1/57, 2%) and absent in South America and the Caribbean (0/40).ConclusionsEPEC and EAEC outnumbered ETEC and Campylobacter everywhere, yet the proportions of pathogen findings varied by region, with ETEC and Campylobacter rates showing the greatest differences. The high frequency of multibacterial findings in many regions indicates a need for further investigation of the clinical role of each pathogen.     

EFFECT OF STRESS ON PRODUCTION OF HEAT LABILE ENTEROTOXIN BY ESCHERICHIA COLI

Enterotoxigenic Escherichia coli (ETEC) is an important pathogen responsible for secretory diarrhoea. The production of heat labile enterotoxin (LT), by ETEC, is largely responsible for the pathogenesis of diarrhoea. In the present study we investigated the effect of stress factors such as temperature, pH, osmotic stress and nutritional limitation on the production of LT by ETEC using in-house GMI-ELISA. Four strains of E. coli consisting, one standard strain MTCC 723 and three clinical isolates were used in the study. Maximum amount of LT (OD 3.285) was produced at 37°C followed by 40°C (OD 3.305). Growth of E. coli in medium with pH 8.6 resulted in maximum amount of LT production (OD 3.489). LT was not detectable when bacteria were grown in medium with pH ≤7.2 and ≥9.2. Sodium chloride concentration of 0.2 M stimulated maximum amount of LT production. Maximum amount of LT was produced when the bacteria were grown in medium containing 2.5g/l of glucose. All the stress factors had a significant effect on the LT production by E. coli, though quantitative differences in the various strains were observed.     

Phenotypic and genotypic characterization of enteroaggregative Escherichia coli isolates from pediatric population in Pakistan

Enteroaggregative Escherichia coli (EAEC) are a leading cause of diarrhea among children. The objective of this study was to define the frequency of EAEC among diarrheal children from flood‐affected areas as well as sporadic cases, determine multidrug resistance, and evaluation of virulence using an in vivo model of pathogenesis. Stool samples were collected from 225 diarrheal children from 2010 to 2011 from flood‐affected areas as well as from sporadic cases in Pakistan. Identified EAEC isolates were characterized by phylogrouping, antibiotic resistance patterns including the extended‐spectrum beta lactamase spectrum, single nucleotide polymorphism detection in gyrA and parC, and virulence potential using wax worm, G. mellonella. A total of 35 (12.5%) confirmed EAEC isolates were identified among 225 E. coli isolates. EAEC isolates displayed high resistance to tetracycline, ampicillin, and cefaclor. A total of 34.28% were ESBL positive. Single nucleotide polymorphism detection revealed 37.14% and 68.57% isolates were positive for SNPs in gyrA (A₆₆₀‐T₆₆₀) and parC (C₃₃₀‐T₃₃₀), respectively. Phylogrouping revealed that B2 phylogroup was more prevalent among all EAEC isolates tested followed by D, A, B1, and non‐typeable (NT). Infection of G. mellonella with EAEC showed that killing infective dose was 100% higher than E. coli DH5 alpha control. EAEC are prevalent among Pakistani children with diarrhea, they are highly resistant to antibiotics, and predominantly fall into B2 phylogroup. Epidemiologic surveillance of EAEC and other E. coli pathotypes is critical to assess not only the role of these pathogens in diarrheal disease but also to determine the extent of multidrug resistance among the population.     

Diarrheagenic Escherichia coli associated with acute diarrhea in children of Cumaná, Venezuela

To establish the prevalence of strains of diarrheagenic Escherichia coli (DEC) associated to acute diarrhea in children of Cumaná, Venezuela, stool samples were taken from 200 children aged < 5 years with acute diarrheal disease, and from 30 healthy children used as control. Isolation and bacterial identification was performed by conventional biochemical tests and stool cultures. The presence of pathogenic genes of each type of DEC was investigated by the technique of polymerase chain reaction (PCR), determining the genes eae and bfp (EPEC), st and lt (ETEC), ipaH and virF (EIEC) Stx1/Stx2 (STEC), aafII (EAEC) and daaE (ADEC). From 169 E. coli, isolates we determined by PCR 10.65% positive for EPEC (1.18% "typical", 9.47% "atypical"); ETEC (5.91%); EAEC (1.78 %), EIEC (0.59%). There were no statistically significant differences regarding the frequency of each "pathotype" in relation to age, but it did occur when related to the sex (p<0.05). The most relevant clinical features were: fever, vomiting and abdominal pain and the greatest percentage of children affected were of the working and marginal classes. These results shown that the strains of DEC are important etiological agents in acute infectious diarrhea in children of Cumaná.     

A single step multiplex PCR for identification of six diarrheagenic E. coli pathotypes and Salmonella

E. coli is generally a commensal but includes some highly pathogenic strains carrying additional genes in plasmids and/or the chromosome. Based on these genes the pathogenic strains are divided into pathotypes including enteropathogenic (EPEC), enterohemorrhagic (EHEC), enterotoxigenic (ETEC), enteroaggregative (EAEC), enteroinvasive (EIEC) and diffusely adherent (DAEC) E. coli. Here, previously developed multiplex PCR strategies for these strains were integrated into one single step multiplex that differentiates all these E. coli pathotypes, usually based on multiple characteristic PCR products. This multiplex PCR works reliably for colony PCR. Two additional markers were added: one to detect most Enterobacteriacea, which acts as a positive control for successful PCR, and one to distinguish Salmonella. The multiplex correctly classified a set of 45 reference strains by colony PCR and 71 (45 + 26) strains by in silico PCR. It was then used to interrogate 44 clinical strains from bovine hosts resulting in detection of EAEC and DAEC determinants.     

Diarrheagenic Escherichia coli and Shigella strains isolated from children in a hospital case-control study in Hanoi, Vietnam

This case-control study detected and characterized Shigella and diarrheagenic Escherichia coli (DEC) types among Vietnamese children less than 5 years old. In 249 children with diarrhea and 124 controls, Shigella spp. was an important cause of diarrhea (P < 0.05). We used multiplex PCR and DNA probes to detect enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAggEC), enteropathogenic E. coli (EPEC), attaching and effacing E. coli (A/EEC), verocytotoxin-producing E. coli (VTEC), and enterotoxigenic E. coli (ETEC). The prevalences of DEC in the diarrhea and control groups were 25.7 and 10.5%, respectively. In 62 children with diarrhea, 64 DEC strains included 22 EAggEC (8.8%), 2 EIEC (0.8%), 23 A/EEC (9.2%), 7 EPEC (2.8%), and 10 ETEC strains (4.0%). Among controls, 13 DEC strains included 5 EAggEC strains (4.0%), 7 A/EEC strains (5.6%), and 1 EPEC strain. The characterization of DEC by serotypes, antimicrobial susceptibility patterns, virulence genes, and pulsed-field gel electrophoresis showed the occurrence of many different and highly heterogenic DEC subtypes, but common serotypes were found among ETEC, EIEC and EPEC, respectively. Serotyping was used to distinguish between A/EEC and EPEC. However, A/EEC, EPEC, and EAggEC were isolated at high frequency from both cases and controls. Further in-depth studies are needed to better understand important virulence factors of DEC, especially A/EEC, EPEC, and EAggEC.     

Controlled study on enteropathogens in travellers returning from the tropics with and without diarrhoea

Diarrhoea is the most frequent health problem among travellers in the tropics. However, data on the spectrum and relevance of enteropathogens in international travellers with and without diarrhoea are limited. Stool samples from 114 cases of diarrhoea in travellers returning from the tropics were collected for microbiological examination and PCR for norovirus genogroups I and II, enteroaggregative Escherichia coli (EAEC), and enterotoxigenic E. coli (ETEC) producing heat-labile toxin (LT) and heat-stable toxin (ST). Travel and laboratory data of cases were compared with those of 56 travellers without diarrhoea. Among cases, EAEC was found in 45% of stool samples, followed by LT-ETEC (20%), ST-ETEC (16%), Blastocystis hominis (15%), Campylobacter jejuni (12%), norovirus (11%), Giardia lamblia (6%), Shigella spp. (6%), and Salmonella spp., Cryptosporidium spp., and Cyclospora cayetanensis (3% each). However, only for EAEC, ST-ETEC, Blastocystis and Campylobacter was the prevalence significantly higher among cases than among controls. Co-infections were common: 61% for cases and 13% for controls. The most common travel destination was Asia (54%), followed by Africa (35%) and Latin America (9%). The highest relative risk for diarrhoea was calculated for travellers to West Africa, East Africa, and South Asia. In this study, EAEC, LT-ETEC and ST-ETEC were detected most frequently in cases of travellers’ diarrhoea. Although enteric infections with EAEC, ST-ETEC and Campylobacter often cause diarrhoea, the pathogenetic relevance remains unclear for most of the other enteropathogens, because of significant prevalence rates also being seen in controls without diarrhoea and the high frequency of co-infections.     

Emerging agents of gastroenteritis: Aeromonas,Plesiomonas, and the diarrheagenic pathotypes of Escherichia coli

Knowledge of the pathogenic roles of certain bacterial agents in gastroenteritis has been growing over the past few decades. With the increasing use of multiplex molecular-based syndromic stool pathogen panels, the roles of Plesiomonas shigelloides and some of the diarrheagenic pathotypes of Escherichia coli (enterotoxigenic E. coli [ETEC], enteropathogenic E. coli [EPEC], enteroinvasive E. coli [EIEC], and enteroaggregative E. coli [EAEC]) have been better understood. Although not currently targeted on Food and Drug Administration (FDA)-cleared commercial multiplex stool panels, Aeromonas has also emerged as a possible cause of bacterial gastroenteritis. The clinical presentation, pathophysiology, and diagnostic approaches to these pathogens in stool specimens are reviewed. Variability in inclusion of these pathogens on multiplex molecular panels and difficulties in detection by stool culture techniques utilized by clinical microbiology laboratories have contributed to an unclear understanding of the pathogenic role of several of these pathogens. Nonetheless, most evidence points towards a clear pathogenic role for P. shigelloides and ETEC, and possibly EPEC and EIEC. The contribution of Aeromonas spp. and EAEC to bacterial gastroenteritis has not been fully established. Further studies of pathogenicity of these pathogens are needed.     

Occurrence and molecular characterization of enteropathogenic Escherichia coli serotypes isolated from children with diarrhoea in Najaf, Iraq

Purpose: Enteropathogenic Escherichia coli (EPEC) are among the most important pathogens infecting children worldwide and are one of the main causes of diarrhoea. The study was carried out to investigate the occurrence of EPEC as a cause of infectious diarrhoea in children younger than 2 years of age and characterize their virulence genes. Materials and Methods: During the study period, a total of 656 faecal specimens from children with diarrhoea and 54 from healthy children were analyzed. E. coli isolates were serotypically identified with EPEC polyvalent and monovalent antisera. The isolated EPEC were examined for the presence of the attaching and effacing (eaeA), bundle-forming pilus (bfpA), Shiga like toxins (stx₁ and stx₂), enterohaemorrhagic E. coli enterohaemolysin (EHEC hlyA) and EPEC adherence factor (EAF) genes by the PCR assay. Results: The study has shown that 22 (3.4%) had diarrhoea due to EPEC, while no EPEC isolates were detected in asymptomatic children. The highest number of the EPEC isolated belonging to polyvalent 2. The primers encoding virulence genes were subjected to all the EPEC isolates. Only 9.1%, 27.3%, and 9.1% isolates gave positive re sults with intimin (eaeA), bfbA and (EAF) genes, respectively. None of the isolates were positive for stx₁, stx₂, and hlyA genes. Typical EPEC (eaeA⁺, bfpA⁺) was diagnosed in two isolates, while, atypical EPEC was manifested in four isolates. Conclusions: According to the results, the frequency of EPEC isolates in Najaf was lower than what has been suspected and the investigation including the use of molecular technique and serotyping, are necessary to allow precise identification and epidemiological study of these pathogens.     

Diarrheagenic Escherichia coli Markers and Phenotypes among Fecal E. coli Isolates Collected from Nicaraguan Infants

We analyzed the prevalence of diarrheagenic Escherichia coli (DEC) markers and common phenotypes in 2,164 E. coli isolates from 282 DEC-positive samples. Enteropathogenic E. coli (EPEC) and enteroaggregative E. coli (EAEC) were very diverse and were not correlated with diarrhea. Enterotoxigenic E. coli (ETEC) estA and enterohemorrhagic E. coli (EHEC) belonged to a few phenotypes and were significantly correlated with diarrhea.In Nicaragua, diarrheagenic Escherichia coli (DEC) strains are considered to be common causes of diarrhea among infants, with enterotoxigenic E. coli (ETEC) and enteropathogenic E. coli (EPEC) the most common DEC types found (4, 6, 12). We previously performed a large screening study on stool samples from 526 infants with and without diarrhea to detect the prevalence of five different categories of DEC and to determine the phenotypic diversity of E. coli isolates from diarrheal and control infants. A one-step multiplex PCR using a mixture of eight primer pairs was applied to the primary streak of E. coli cultures, and eight isolates per sample were analyzed by biochemical fingerprinting (7, 12). As many as 54% of the diarrheal samples and 53% of the control samples were positive for one or more DEC markers, and no clear connection between biochemical phenotypes found in DEC-positive samples and diarrhea could be established.For the present report, we analyzed eight E. coli isolates from each of the 282 DEC-positive stool samples by PCR for the occurrence of the respective DEC markers found in the primary streak.     

Genetic detection of diarrheagenic Escherichia coli isolated from children with sporadic diarrhea.

Escherichia coli strains are among the major bacterial causes of diarrheal illness. At least 5 categories of diarrheagenic E. coli (DEC) are recognized, namely enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), and enterohemorrhagic E. coli (EHEC). Due to the need for costly and labor-intensive diagnostic procedures, identification of DEC is difficult at standard laboratories. Therefore, the epidemiology of DEC infections remains obscure in Taiwan. Recently, polymerase chain reaction (PCR) or dot blot has been used for genetic detection of DEC. In this study, we analyzed 150 E. coli isolates from diarrheal stools of children under 5 years old. The PCR tests detected 5 ETEC (3.3%), 6 EPEC (4%), 4 EIEC (2.7%), and 13 EAEC (8.7%) isolates. No EHEC was detected. Dot blot and sequence analysis were used to confirm the results of PCR. The cellular fatty acid (CFA) profiles from E. coli isolates were also analyzed. Comparison of CFA composition revealed minor variation in the percentage of each fatty acid detected among DEC isolates of ETEC, EPEC, EIEC and EAEC, but did not provide enough evidence for differentiating between categories of DEC by CFA profiles alone.     

Group A rotavirus and bacterial agents associated with diarrhoea-induced hospitalisations in children below 5 years of age in Jammu

Out of 210 faecal samples collected from children below 5 years attending different hospitals in Jammu and exhibiting clinical signs of diarrhoea, 41.9% samples were found positive for group A rotavirus by RNA-PAGE. Escherichia coli isolated in the study belonged to nine serogroups, out of which O69 was most frequent, being present in 12.38% samples. E. coli serogroups well recognised as enteropathogens viz. O69, O20 and O153 were present in 27.6% samples. Other bacterial pathogens associated with diarrhoea were present in 8.09% samples, out of which Shigella spp. was found in 4.76% samples followed by Salmonella spp. (2.38%) and Pseudomonas spp. (0.95%).     

High level Multi-Resistant and Virulent Escherichia coli in Abeokuta,Nigeria

Multi-resistant Escherichia coli (E. coli) strains co-harboring virulence genes is a cause of high morbidity in Abeokuta, Nigeria. This study was designed to determine some virulent factors among enteropathogenic E. coli in Abeokuta, Nigeria. Approximately non-repetitive 102 isolates of E. coli were recovered from clinical samples from two health facilities in Abeokuta. Biotyping using API and antibiotic susceptibility was determined, and eae and flic genes were assayed by PCR. Antibiotic resistance relatedness was performed by DendroUPGMA. Results showed that 48.0% and 52.0 % were intestinal and extra-intestinal E. coli, ampicillin recorded 100% resistance, amoxycilli/clavulanic acid 64.7%, cotrimoxazole 57.8% and 56.8% resistance against cefotaxime, at MIC >16 ug/mL, 100%, 57.8%, and 50% have MIC50 to ampicillin, tetracycline, and ceftazidime, while 74.5% and 48.0% have MIC90 to ampicillin and ceftazidime. Significant rates of 4.9%, 7.8%, and 9.8% flic, eae, and flic/eae genes were found in intestinal isolates, while 2.9%, 2.0%, and 3.9% were found in extra-intestinal (P < 0.05). Two major clades of the resistant isolates reveal significant antibiotic relatedness among intestinal and extra-intestinal isolates, at 54% resistance similarities with very high multi-antibiotic resistance index of 1.0 (MARI). A high rate of undetected virulent E. coli pathotypes with high resistance could trigger unprecedented morbidity and mortality, mostly among children and the elderly.     

Enteroaggregative Escherichia coli Heat-Stable Enterotoxin 1 (EAST1): A New Toxin with an Old Twist

Enteroaggregative Escherichia coli heat-stable enterotoxin 1 (EAST1) is a small protein that was first detected more than a decade ago in an enteroaggregative E. coli (EAEC) strain isolated from the stools of a diarrheic child. The EAST1 gene, astA, is not solely present in EAEC, but also in other categories of diarrheagenic E. coli. Strains expressing EAST1 have been shown to induce diarrhea principally in humans, although they have also been associated with piglets and calves. EAST1 toxin has been proposed as a virulence factor implicated in the mechanism of pathogenesis of EAEC and could play a role in the pathogenicity of other enteropathogens as well. This toxin is often compared to E. coli STa enterotoxin because they share some physical and mechanistic similarities. This review summarizes the various observations on EAST1 since its discovery.