ETEC Strains Research Articles

Detection of pathogenic Escherichia coli and microbiological quality of chilled shrimp sold in street markets.

Foodborne illnesses caused by Escherichia coli are one of the most important gastrointestinal diseases and therefore represent a public health risk. The presence of E.coli in water or in products such as shrimp indicates faecal contamination. However, indicator micro-organisms can be used to evaluate the microbiological quality of food sold in markets. This study focused on detecting isolates of E.coli containing the genes stx1A, stx2A, eae, LTI, STa, STb, aggR and pCVD432 in chilled shrimp sold in street markets in the municipality of São Paulo, Brazil, and to assess the microbiological quality of this product. Enteropathogenic and enterotoxigenic E.coli pathotypes were detected on the surface of two chilled shrimp samples. Salmonella spp. was not isolated. In addition, contamination of surface and muscle of the shrimp samples was found to be correlated. The detection of EPEC and ETEC pathotypes in chilled shrimp sold in street markets in Brazil provides useful epidemiological information for public health authorities to improve food safety and public health. Shrimps are crustaceans commonly produced and consumed in Brazil. Specimens of Farfantepenaeus brasiliensis and Litopenaeus schmitti sold in street markets were examined by PCR to detect the presence of Escherichia coli pathotypes (enteropathogenic, enterotoxigenic, enterohemorrhagic and enteroinvasive). EPEC and ETEC strains were detected in whole shrimp. These findings provide useful information for public health authorities to improve the food safety and health of the Brazilian population.

Towards Rational Design of a Toxoid Vaccine against the Heat-Stable Toxin of Escherichia coli.

Enterotoxigenic Escherichia coli(ETEC) is an important cause of diarrheal disease and death in children <5 years old. ETEC strains that express the heat-stable toxin (ST), with or without the heat-labile toxin, are among the four most important diarrhea-causing pathogens. This makes ST an attractive target for an ETEC vaccine. An ST vaccine should be nontoxic and elicit an immune response that neutralizes native ST without cross-reacting with the human endogenous guanylate cyclase C receptor ligands. To identify variants of ST with no or low toxicity, we screened a library of all 361 possible single-amino-acid mutant forms of ST by using the T84 cell assay. Moreover, we identified mutant variants with intact epitopes by screening for the ability to bind neutralizing anti-ST antibodies. ST mutant forms with no or low toxicity and intact epitopes are termed toxoid candidates, and the top 30 candidates all had mutations of residues A14, N12, and L9. The identification of nontoxic variants of L9 strongly suggests that it is a novel receptor-interacting residue, in addition to the previously identified N12, P13, and A14 residues. The screens also allowed us to map the epitopes of three neutralizing monoclonal antibodies, one of which cross-reacts with the human ligand uroguanylin. The common dominant epitope residue for all non-cross-reacting antibodies was Y19. Our results suggest that it should be possible to rationally design ST toxoids that elicit neutralizing immune responses against ST with minimal risk of immunological cross-reactivity.

Prophylaxis of post-weaning diarrhoea in piglets by zinc oxide and sodium humate

The high prophylactic doses of ZnO commonly used to control post-weaning diarrhoea (PWD) in piglets have become an environmental problem. The possibility of partial replacement of ZnO by sodium humate (HNa) in PWD treatment was investigated in this study. Weaned piglets (32) were challenged with two enterotoxigenic E. coli strains (ETEC/O149/F4/LT and ETEC/O147/F18/LT) and allocated into four treatments maintained for three weeks: C (no supplementation); ZnO2.5 (2.5 g ZnO/kg diet); HNa + ZnO1.0; HNa + ZnO1.5; HNa + ZnO1.7 (20 g HNa and 1.0, 1.5 and 1.7 g ZnO/kg diet, respectively). The effects on incidence, severity and duration of diarrhoea, faecal shedding of total E. coli and both ETEC strains, growth rate and selected blood parameters were investigated. In contrast with ZnO2.5 and HNa + ZnO1.7, high daily diarrhoea scores, incidence and duration and mortality due to severe dehydration were seen in C, HNa + ZnO1.0 and HNa + ZnO1.5 groups. The administration of ZnO and HNa did not affect the faecal shedding of the challenged ETEC strains for eght days, even in clinically healthy piglets in ZnO2.5 and HNa + ZnO1.7 groups. Signs of growth depression were found in C; HNa + ZnO1.0 and HNa + ZnO1.5 groups during the first week. No difference in growth performance was observed in ZnO2.5 and HNa + ZnO1.7 piglets. Most of the selected biochemical and haematological parameters did not differ significantly among the treatments. However, a significantly higher serum Zn as a result of high dietary ZnO intake in the ZnO2.5 group compared to the control and HNa groups was detected. Significantly lower serum P in ZnO2.5; HNa + ZnO1.7 and HNa + ZnO1.0 groups compared to the control group was most likely induced by the increased Zn in serum. The results indicate the possibility of reducing the high pharmacological levels of ZnO in the prophylaxis of PWD through partial replacement with HNa. Such a treatment maintains the favourable prophylactic effect while lowering the Zn content in faeces.

A small-scale experiment of using phage-based probiotic dietary supplement for prevention of E. coli traveler's diarrhea.

Traveler's diarrhea (TD) is caused by Escherichia coli in 30% of cases. We have developed a phage cocktail for prophylaxis of TD caused by E.coli, Shigella flexneri, Shigella sonnei, Salmonella enterica, Listeria monocytogenes or Staphylococcus aureus, and investigated its effectiveness against infection caused by the non-pathogenic Lac (-) strain of E.coli K12 C600 in animal and human trials. On the 6thday of both animal and human trials E. coli K12 C600 strain was detected in titer of 104 CFU/g of mice feces and 106 CFU/g of human feces in the control (untreated) groups, while it was not detected in the samples of either of the study (phage-treated) groups. These results have great significance because the original coliphages included in the cocktail have a broad host-range including ETEC, EAEC and EHEC strains which cause severe cases of TD.

Molecular screening of pathogenic Escherichia coli strains isolated from dairy neonatal calves in Cordoba province, Argentina

The aim of this study was to perform a current molecular characterization of bovine pathogenic Escherichia coli strains isolated from random samplings in Argentinean dairy farms. Rectal swabs were obtained from 395 (63.7%) healthy and 225 (36.3%) diarrheic calves, belonging to 45 dairy farms in Cordoba Province, Argentina. E. coli isolates were examined for virulence genes (f5, f41, f17, sta, stb, lt, eae, vt) using PCR and the prevalence of E. coli virulence profiles was spatially described in terms of spatial distribution. A total of 30.1% isolates were found to be positive for at least one of the virulence genes. Depending on the different gene combinations present, 11 virulence profiles were found. Most of the isolates analyzed had a single gene, and no combination of fimbrial and enterotoxin gene was predominant. There was no association between the frequency and distribution of E. coli virulence genes and calf health status. Most of the virulence profiles were compatible with ETEC strains and showed a homogeneous distribution over the sampled area. A clustering pattern for E. coli virulence profiles could not be recognized. This work provides updated information on the molecular characterization of pathogenic E. coli strains from dairy herds in Cordoba, Argentina. These findings would be important to formulate prevention programs and effective therapies for diarrhea in calves caused by E. coli.

English

INTRODUCTION: Diarrhoea is defined as passage of loose, liquid or watery stools. In the developing countries, diarrhoea remains a wet season disease with bacteria playing a greater role. The agents responsible for infantile diarrhoea may be bacteria, viruses, parasites, malnutrition and drugs. Infectious agents that cause diarrhoeal disease are usually spread by the fecal-oral route. Escherichia coli (E. coli) is a gram-negative bacillus that may be found in the normal intestinal flora of humans and animals, but can also be an important cause of enteric illness. Our present study is an attempt to fulfil this vacuum and investigate the most common pathogens causing these diseases amongst patients in and around a tertiary care hospital of eastern Bihar, with special reference to the serotypes of Escherichia coli. MATERIAL AND METHODS: Samples were collected from both male and female patients below five years of age, admitted to the Pediatrics Department, with complains of diarrhoea and gastroenteritis. Samples were processed, identified and antibiotic sensitivity was done as per standard protocol. Serotyping of E. coli isolates was carried out using antisera as per manufacturer's instructions. RESULTS: Different diarrhoeagenic strains of E. coli showed that the maximum numbers of strains were EPEC, followed by DAEC. EAEC and ETEC accounted for only a small percentage of strains. Upon Serotyping the different E. coli isolates from patients with diarrhoea, the results showed that the Serotype O26, O111 and O55 were the predominant serotypes of EPEC. Among the ETEC strains only 2 serotypes were seen viz. O6 and O15. Serotype O44 and O125 were the only two serotypes of EAEC seen. Among the DAEC, the only serotype was O1. The antibiotic susceptibility pattern of various isolates shows maximum resistance against ampicillin followed by cefixime. Strains were significantly sensitive to amikacin, gentamicin and ofloxacin. CONCLUSION: Our result shows high rate of diarrhoeagenic E. coli among Indian children presenting with diarrhoea particularly in Katihar / Kosi region of Bihar. The finding of diverse E. coli subtypes even amongst the small number of E. coli isolates highlights the importance of pathogenic E. coli in cases of diarrhoea and gastroenteritis and stresses upon the need for enhanced surveillance of children with diarrhoea in these parts of the globe.

Simultaneous exposure to Escherichia coli heat-labile and heat-stable enterotoxins increases fluid secretion and alters cyclic nucleotide and cytokine production by intestinal epithelial cells.

Enterotoxigenic Escherichia coli (ETEC) is a significant cause of diarrheal disease and death, especially in children in developing countries. ETEC causes disease by colonizing the small intestine and producing heat-labile toxin (LT), heat-stable toxin (ST), or both LT and ST (LT+ST). The majority of ETEC strains produce both ST and LT. Despite the prevalence of LT+ST-producing organisms, few studies have examined the physiologic or immunologic consequences of simultaneous exposure to these two potent enterotoxins. In the current report, we demonstrate that when LT and ST are both present, they increase water movement into the intestinal lumen over and above the levels observed with either toxin alone. As expected, cultured intestinal epithelial cells increased their expression of intracellular cyclic GMP (cGMP) when treated with ST and their expression of intracellular cyclic AMP (cAMP) when treated with LT. When both toxins were present, cGMP levels but not cAMP levels were synergistically elevated compared with the levels of expression caused by the corresponding single-toxin treatment. Our data also demonstrate that the levels of inflammatory cytokines produced by intestinal epithelial cells in response to LT are significantly reduced in animals exposed to both enterotoxins. These findings suggest that there may be complex differences between the epithelial cell intoxication and, potentially, secretory outcomes induced by ETEC strains expressing LT+ST compared with strains that express LT or ST only. Our results also reveal a novel mechanism wherein ST production may reduce the hosts' ability to mount an effective innate or adaptive immune response to infecting organisms.

Characterization of immunological cross-reactivity between enterotoxigenic Escherichia coli heat-stable toxin and human guanylin and uroguanylin.

Enterotoxigenic Escherichia coli (ETEC) expressing the heat-stable toxin (ST) (human-type [STh] and porcine-type [STp] variants) is among the five most important enteric pathogens in young children living in low- and middle-income countries. ST mediates diarrheal disease through activation of the guanylate cyclase C (GC-C) receptor and is an attractive vaccine target with the potential to confer protection against a wide range of ETEC strains. However, immunological cross-reactivity to the endogenous GC-C ligands guanylin and uroguanylin is a major concern because of the similarities to ST in amino acid sequence, structure, and function. We have investigated the presence of similar epitopes on STh, STp, guanylin, and uroguanylin by analyzing these peptides in eight distinct competitive enzyme-linked immunosorbent assays (ELISAs). A fraction (27%) of a polyclonal anti-STh antibody and an anti-STh monoclonal antibody (MAb) cross-reacted with uroguanylin, the latter with a 73-fold-lower affinity. In contrast, none of the antibodies raised against STp, one polyclonal antibody and three MAbs, cross-reacted with the endogenous peptides. Antibodies raised against guanylin and uroguanylin showed partial cross-reactivity with the ST peptides. Our results demonstrate, for the first time, that immunological cross-reactions between ST and the endogenous peptides can occur. However, the partial nature and low affinity of the observed cross-reactions suggest that the risk of adverse effects from a future ST vaccine may be low. Furthermore, our results suggest that this risk may be reduced or eliminated by basing an ST immunogen on STp or a selectively mutated variant of STh.

A study of the prevalence of diarrhoeagenic Escherichia coli in children from Gwagwalada, Federal Capital Territory, Nigeria.

IntroductionDiarrhoeagenic Escherichia coli (DEC) are major causes of diarrhoea in Nigeria. This study was conducted to determine the prevalence of diarrhoea caused by DEC within the Federal Capital Territory, Abuja, Nigeria.MethodsA total of 730 rectal swabs obtained from 201 children with diarrhoea and 529 healthy controls aged 0-24 months were cultured for the isolation of Escherichia coli. All E. coli isolates were investigated by PCR to determine their pathotype.ResultsA total of 61 DEC strains were recovered at a rate of 18.4% and 2.6% from children with diarrhoea and healthy controls respectively. The DEC strains recovered were Enteroaggregative Escherichia coli (34.4%), Shiga-toxin producing Escherichia coli (31.1%), Enterotoxigenic Escherichia coli(18.0%), typical enteropathogenic Escherichia coli (15.0%) and Enteroinvasive Escherichia coli (1.6%). Shiga-toxin producing Escherichia coli andEnteroinvasive Escherichia coli were recovered only from children suffering from diarrhoea and the overall prevalence of DEC strains was significantly higher among the children with diarrhoea (P < 0.0001). The number of DEC strains obtained during the dry season was significantly higher (P = 0.012) than the number obtained in the rainy season.ConclusionDiarrhoea caused by E. coli in the Nigerian children studied is associated with several diarrhoeagenic pathotypes and a significant proportion of the healthy children were found to harbour EAEC and ETEC strains. These asymptomatic carriers may be regarded as potential transmitters of infection to vulnerable children in the study area.

English

Six lytic bacteriophages (JH1-JH6) lytic against O8 ETEC were isolated from 40 individual fecal samples and 30 sewage samples from pig farms in Nanjing, China. Two of the bacteriophages (JH1 and JH2) with icosahedral heads, necks, contractile tails belonged to the&nbsp;Myoviridae&nbsp;family.&nbsp;The other four bacteriophages (JH3, JH4, JH5 and JH6) had relatively thin, long, non-contractile and belonged to the&nbsp;Siphoviridae&nbsp;family. Unlike typicalSiphoviridae&nbsp;family members&rsquo; morphologyes had flexible tails, whereas JH5 had the straight tails and fibers. All bacteriophages produced clear plaques in double agar, with JH6 producing the largest plaques. Phages JH1 to JH6 lysed 50 to 100% of 12 O8: F18 ETEC strains, 50 to 75% of 20 O8:F4 ETEC, 5 to 42.5% of 40 non-O8 ETEC and 4.76 to 9.52% of 21 chickens&nbsp;E. coli&nbsp;strains. No lysis of&nbsp;E. coli&nbsp;strain from cows, as well asEscherichia coli&nbsp;in other O serogroups (O2, O5, O9, O11, O138, O139, O141 and O149), was observed. All bacteriophages titers were unaffected by exposure to pH 5 to 9 for 16 h. Sodium dodecyl sulphate-polyacrilamide gel electrophoresis (SDS-PAGE) obtained from proteins was extracted from the various bacteriophages. The proteomes of JH1 and JH2 appear to be identical while that of the other four phages are all different indicating they are probably different.&nbsp;These six bacteriophages are potential candidates for prophylaxis and therapeutic strategies against diarrhea in piglets caused by O8 ETEC. &nbsp; Key words:&nbsp;O8 serogroup, bacteriophage, enterotoxigenic,&nbsp;Escherichia coli, diarrhea in piglets.

Adhesion of enterotoxigenic Escherichia coli strains to neoglycans synthesised with prebiotic galactooligosaccharides

Enterotoxigenic (ETEC) Escherichia coli (E. coli) causes traveller’s diarrhoea and high mortality among baby animals. ETEC adhesion is mediated by lectins (adhesins) that bind to glycoconjugates on the surface of host cells. Glycans that compete for adhesion could be used for disease prevention. Neoglycans of porcine albumin (PSA) that were conjugated with prebiotic galactooligosaccharides (GOS) were synthesised using the Maillard reaction. PSA glycation was confirmed by a reduction in the number of available free amino groups, decreased tryptophan intrinsic fluorescence, increased molecular mass and Ricinus communis lectin recognition. The adhesion of four ETEC strains (E. coli H10407, CFA+, K99 and K88) to PSA–GOS was examined by an enzyme-linked lectin assay. E. coli K88 bound to PSA–GOS with greater affinity (P<0.05) than did E. coli H10407, CFA+ and K99. In addition, PSA–GOS partially inhibited the adherence of the K88 strain to intestinal mucins. Pig ETEC strain was unable to ferment galactooligosaccharide–neoglycans. These results suggest that neoglycans obtained by the Maillard reaction may serve in the prophylaxis of ETEC K88 diarrhoea.

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.

Antibiotic resistance patterns of STEC and ETEC strains: A study on frozen foods of animal origin and children with acute diarrhea

Amac: Shigatoxin ureten E. coli (STEC) ve Enterotoksijenik E. coli (ETEC) tum dunyada gida ve su kaynakli insan hastaliklarindan sorumlu tutulan enfeksiyoz patojenlerdir. Bu calismanin amaci, STEC ve ETEC suslarinin insidansini belirlemek ve antibiyotik direnc paternlerini degerlendirmektir.Yontemler: E. coli suslarini izole edilmesi ve standart prosedurler esas alinarak tanimlanmasi icin hayvansal kokenli toplam 125 dondurulmus gida ve akut ishali olan cocuklardan 466 rektal suruntu alindi. E. coli suslarinin antimikrobiyal duyarlilik testleri Klinik Laboratuvar Standartlari Enstitusu’ne gore yapildi. STEC ve ETEC suslarinda iki ya da daha fazla antimikrobiyal gruba karsi direnc varligi kaydedildi.Bulgular: Akut ishali olan cocuklardan alinan 466 rektal suruntu orneginden toplam 87 E. coli susu ve 125 dondurulmus hayvansal kokenli gida orneginden 40 E. coli susu tespit edildi. Test sonuclari hayvansal kokenli dondurulmus gidalardan izole edilen E. coli suslarindaki Shiga toksin (stx) ve enterotoksin (estA) genlerinin prevalansini %5,0 ve %2,5 olarak gosterdi. Benzer sekilde, cocuklardan izole edilen E. coli suslarinda Shiga toksin (stx) ve enterotoksin (estA) genlerinin prevalansi ise %5,7 ve %4,5 idi.Sonuc: Shiga toksin (stx), ve enterotoksin (estA) genleri bulunduran E. coli suslarinin, Sanandaj icin potansiyel bir enfeksiyon kaynagi olmadigi sonucuna vardik; belki de gida kaynakli hastaliklarin en onemli nedeni diger enterik patojenlerdir

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.

Verotoxin -producing Escherichia coli Old Bug New Infections

EnteropathogenicEscherichia coli first discovered in the last century from the fecal flora of neonates.E.coli resides in human and animal intestine as normal flora.ETEC,EPEC and,EHEC strains cause severe potentially fatal disease . ETEC strains are important cause of childhood diarrhea in developing countries .EPEC infections are acquired by person to person and in the hospitals .EHECs Shiga producing strains that induce bloody diarrhea lead to HUS include renal failure microangiopathic haemolyticanaemia and thrombocytopenia.Vero toxin (Shiga like toxin) can directly damage renal and endothelial cells. The reservoir of EHEC strains is the gastrointestinal tract of young cattle and herbivorous mammals. Low infectious dose of EHEC strains facilitates the transmission of infection. Treatment of EHEC with antimicrobials have not shown clinical benefit rather cause increased production of toxins. Prevention of TD consists to avoid dietary indiscretions, hand washing before eating and eating only cooked food in endemic countries.

Differential gene expression profiling of porcine epithelial cells infected with three enterotoxigenic Escherichia coli strains

BackgroundEnterotoxigenic Escherichia coli (ETEC) is one of the most important pathogenic bacteria causing severe diarrhoea in human and pigs. In ETEC strains, the fimbrial types F4 and F18 are commonly found differently colonized within the small intestine and cause huge economic losses in the swine industry annually worldwide. To address the underlying mechanism, we performed a transcriptome study of porcine intestinal epithelial cells (IPEC-J2) with and without infection of three representative ETEC strains.ResultsA total 2443, 3493 and 867 differentially expressed genes were found in IPEC-J2 cells infected with F4ab ETEC (CF4ab), with F4ac ETEC (CF4ac) and with F18ac ETEC (CF18ac) compared to the cells without infection (control), respectively. The number of differentially expressed genes between CF4ab and CF4ac, CF4ab and CF18ac, and CF4ac and CF18ac were 77, 1446 and 1629, respectively. The gene ontology and pathway analysis showed that the differentially expressed genes in CF4abvs control are significantly involved in cell-cycle progress and amino acid metabolism, while the clustered terms of the differentially expressed genes in CF4acvs control comprise immune, inflammation and wounding response and apoptosis as well as cell cycle progress and proteolysis. Differentially expressed genes between CF18acvs control are mainly involved in cell-cycle progression and immune response. Furthermore, fundamental differences were observed in expression levels of immune-related genes among the three ETEC treatments, especially for the important pro-inflammatory molecules, including IL-6, IL-8, TNF-α, CCL20, CXCL2 etc.ConclusionsThe discovery in this study provides insights into the interaction of porcine intestinal epithelial cells with F4 ETECs and F18 ETEC, respectively. The genes induced by ETECs with F4 versus F18 fimbriae suggest why ETEC with F4 may be more virulent compared to F18 which seems to elicit milder effects.

Identification of Coli Surface Antigen 23, a Novel Adhesin of Enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea, mainly in developing countries. Although there are 25 different ETEC adhesins described in strains affecting humans, between 15% and 50% of the clinical isolates from different geographical regions are negative for these adhesins, suggesting that additional unidentified adhesion determinants might be present. Here, we report the discovery of Coli Surface Antigen 23 (CS23), a novel adhesin expressed by an ETEC serogroup O4 strain (ETEC 1766a), which was negative for the previously known ETEC adhesins, albeit it has the ability to adhere to Caco-2 cells. CS23 is encoded by an 8.8-kb locus which contains 9 open reading frames (ORFs), 7 of them sharing significant identity with genes required for assembly of K88-related fimbriae. This gene locus, named aal (adhesion-associated locus), is required for the adhesion ability of ETEC 1766a and was able to confer this adhesive phenotype to a nonadherent E. coli HB101 strain. The CS23 major structural subunit, AalE, shares limited identity with known pilin proteins, and it is more closely related to the CS13 pilin protein CshE, carried by human ETEC strains. Our data indicate that CS23 is a new member of the diverse adhesin repertoire used by ETEC strains.

Distribution of Classical and Nonclassical Virulence Genes in Enterotoxigenic Escherichia coli Isolates from Chilean Children and tRNA Gene Screening for Putative Insertion Sites for Genomic Islands

Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea. Three adhesins (Tia, TibA, EtpA), an iron acquisition system (Irp1, Irp2, and FyuA), a GTPase (LeoA), and an autotransporter (EatA) are ETEC virulence-related proteins that, in contrast to the classical virulence factors (enterotoxins and fimbrial colonization factors) have not heretofore been targets in characterizing isolates from epidemiological studies. Here, we determined the occurrence of these nonclassical virulence genes in 103 ETEC isolates from Chilean children with diarrhea and described their association with O serogroups and classical virulence determinants. Because tia, leoA, irp2, and fyuA are harbored by pathogenicity islands inserted into the selC and asnT tRNA genes (tDNAs), we analyzed the regions flanking these loci. Ten additional tDNAs were also screened to identify hot spots for genetic insertions. Associations between the most frequent serogroups and classical colonization factor (CF)-toxin profiles included O6/LT-STh/CS1-CS3-CS21 (i.e., O6 serogroup, heat-labile [LT] and human heat-stable [STh] enterotoxins, and CFs CS1, -3 and -21), O6/LT-STh/CS2-CS3-CS21, and O104-O127/STh/CFAI-CS21. The eatA and etpA genes were detected in more than 70% of the collection, including diverse serogroups and virulence profiles. Sixteen percent of the ETEC strains were negative for classical and nonclassical adhesins, suggesting the presence of unknown determinants of adhesion. The leuX, thrW, and asnT tDNAs were disrupted in more than 65% of strains, suggesting they are hot spots for the insertion of mobile elements. Sequences similar to integrase genes were identified next to the thrW, asnT, pheV, and selC tDNAs. We propose that the eatA and etpA genes should be included in characterizations of ETEC isolates in future epidemiological studies to determine their prevalence in other geographical regions. Sequencing of tDNA-associated genetic insertions might identify new ETEC virulence determinants.

Bacteriological and physico-chemical assessment of wastewater in different region of Tunisia: impact on human health

BackgroundIn many parts of the world, health problems and diseases have often been caused by discharging untreated or inadequately treated wastewater. In this study, we aimed to control physico-chemical parameters in wastewater samples. Also, microbiological analyses were done to reveal Salmonella strains and each Escherichia coli (E.coli) pathotype.FindingsSixty wastewater samples were collected from fifteen different regions of Tunisia. All physico-chemical parameters (pH, residual free chlorine, total suspended solids, biological oxygen demand, and chemical oxygen demand) were evaluated.For microbiological analyses, samples were filtered to concentrate bacteria. DNA was extracted by boiling and subjected to polymerase chain reaction (PCR) using different pairs of primers.The mean pH values recorded for the sampling point were above the WHO pH tolerance limit. The total suspended solids (TSS) concentrations varied between 240 mg/L and 733 mg/L in entrance points and between 13 mg/L and 76 mg/L in exit points. In entrance points, the studied wastewater has an average COD concentration that varied between 795 mg/mL to 1420 mg/mL. Whereas, BOD concentration of the wastewater ranged between 270 mg/L to 610 mg/L. In exit points, COD concentration varied between 59 mg/L and 141 mg/L, whereas BOD concentration ranged from 15 mg/L to 87 mg/L.The bacteriological control of wastewaters showed that, in entrance points, Escherichia coli (E.coli) was detected at the rate of 76.6%. Three E.coli pathotypes were found: ETEC (53.3%), EAEC (16.6%) and EIEC (6.6%).Concerning the ETEC isolated strains, 8 of 16 (50%) have only the heat-labile toxin gene, 5 of 16 (31.2%) present only the heat-stable toxin gene and 3 of 16 (18.7%) of strains possess both heat-labile toxin gene and heat-stable toxin gene. In exist point, the same pathotypes were found but all detected ETEC strains present only the "est" gene.Concerning Salmonella isolated strains; percentages of 66.6% and 20% were found in entrance and exit points respectively.ConclusionsWastewaters contain a large amount of pathogenic bacteria that present a real impact on human health. Assessment wastewater treatment stations have to consider in account enterobacterial pathogens as potential pathogens that should be correctly controlled.

Detection of stx1, stx2, LT and ST toxin genes and O157 and H7 antigen genes among uropathogenic Escherichia coli isolates from Iran

Infection with Shiga like toxin-producing strains of Escherichia coli (STEC) in addition to diarrhea is associated with severe and fatal complications such as hemolytic uremia syndrome (HUS). Nonetheless, the conventional diagnostic methods of the strains are not precise, and accurate data regarding their infection incidence is not available in many countries including Iran. The aim of this study was to detect stx1, stx2, LT and ST toxin genes, as well as O157 and H7 antigen genes in E. coli samples isolated from patients with urinary tract infection. A total of 100 urinary samples were collected from patients with urinary tract infection who were referring to the health centers in Tehran, Iran, during years 2010-2011. E. coli isolation was performed according to the standard laboratory methods.DNA was extracted from samples and the presence of stx1, stx2, lt, st, O157 and H7genes was investigated by PCR method. Out of 100 samples, 10 (10%) were carryingstx1, 7 (7%) H7, 6 (6%) stx2, 2 (2%) lt, and 1 (1%) O157 genes. One sample was carrying both stx1 and stx2 genes, while none of samples were carrying st gene. The results indicate that ETEC and STEC strains, with a relatively conserved genetic pool, are capable of causing urinary tract infection. Due to the importance of these strains in public health, it is suggested that the conventional methods used for their detection be replaced by a molecular method especially based on the presence of stx genes. Key words: Escherichia coli, urinary tract infections, stx1, stx2, LT toxin gene, ST toxin gene, O157 antigen gene, H7 antigen genes.