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The insertion of the DNA sequence encoding SKIK peptide adjacent to the M start codon of a difficult-to-express protein enhances protein production in Escherichia coli. In this report, we reveal that the increased production of the SKIK-tagged protein is not due to codon usage of the SKIK sequence. Furthermore, we found that insertion of SKIK or MSKIK just before the SecM arrest peptide (FSTPVWISQAQGIRAGP), which causes ribosomal stalling on mRNA, greatly increased the production of the protein containing the SecM arrest peptide in the E. coli-reconstituted cell-free protein synthesis system (PURE system). A similar translation enhancement phenomenon by MSKIK was observed for the CmlA leader peptide, a ribosome arrest peptide, whose arrest is induced by chloramphenicol. These results strongly suggest that the nascent MSKIK peptide prevents or releases ribosomal stalling immediately following its generation during the translation process, resulting in an increase of protein production., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Using multiplex real-time PCR, 960 fecal samples collected from poultry, cattle, and patients with diarrhea in Bangladesh were screened for diarrheagenic Escherichia coli (DEC). The invasion-related gene virB showed the highest prevalence in human patients (41%) and was shown to be positively correlated first with afaB with regards to diffuse adhesion and second with aggR with regards to aggregative adhesion. These three genes were specific to human patients. In contrast, the Shiga toxin genes stx1 (57%) and stx2 (40%) were prevalent in cattle samples. The eae gene, which is associated with attaching and effacing lesion formation, and the elt and est genes, which are associated with enterotoxins, were detected from all three sample sources. Heat map construction and hierarchical clustering assigned the samples into five different clusters, with the patient samples positive for virB and afaB being placed together in one cluster. Although the detection of virulence genes cannot be a direct indication of the distribution of diarrheagenic organisms, their detection suggests that Shigella spp. or enteroinvasive E. coli are the most prevalent diarrheagenic bacteria in Bangladesh and that diffusely adherent E. coli is concomitantly present with these bacteria. eae-possessing organisms in patients may come from cattle and poultry sources. The small number of stx-positive patients could be explained by the small number of animal samples that were positive for both eae and stx.

Diarrheagenicity of diffusely adherent Escherichia coli (DAEC) remains controversial. Previously, we found that motile DAEC strains isolated from diarrheal patients induced high levels of interleukin 8 (IL-8) secretion via Toll-like receptor 5 (TLR5). However, DAEC strains from healthy carriers hardly induced IL-8 secretion, irrespective of their possessing flagella. In this study, we demonstrated that SK1144, a DAEC strain from a healthy carrier, suppressed IL-8 and IL-6 secretion from human epithelial cell lines. Suppression of IL-8 in human embryonic kidney (HEK293) cells that were transformed to express TLR5 was observed not only upon inflammatory stimulation by flagellin but also in response to tumor necrosis factor alpha (TNF-α) and phorbol myristate acetate (PMA), despite the fact that the TNF-α- and PMA-induced inflammatory pathways reportedly are not TLR5 mediated. SK1144 neither decreased IL-8 transcript accumulation nor increased intracellular retention of IL-8. No suppression was observed when the bacteria were cultured in Transwell cups above the epithelial cells; however, a nonadherent bacterial mutant (lacking the afimbrial adhesin gene) still inhibited IL-8 secretion. Direct contact between the bacteria and epithelial cells was necessary, but diffuse adhesion was dispensable for the inhibitory effects. Infection in the presence of chloramphenicol did not suppress cytokine release by the epithelial cells, suggesting that suppression depended on effectors synthesized de novo Inflammatory suppression was attenuated with infection by a bacterial mutant deleted for hcp (encoding a component of a type VI secretion system). In conclusion, DAEC strains from healthy carriers impede epithelial cell cytokine secretion, possibly by interfering with translation via the type VI secretion system., (Copyright © 2018 American Society for Microbiology.)

The source and routes of diarrheagenic Escherichia coli (DEC) remain poorly understood. To investigate the involvement of domestic animals in the dissemination of DEC, the prevalence of DEC in foods and fecal specimens from cattle, pigs, chickens, healthy carriers, and patients in Osaka and Hyogo, Japan was investigated using a multiplex real-time Polymerase Chain Reaction assay. The most abundant virulence genes were astA and eae, which had a prevalence 46.8% and 27.4%, respectively. Additionally, stx1 (26.6%) and stx2 (45.9%) were prevalent in cattle feces, while est (8.5%) and elt (7.6%) were prevalent in pig feces. afaB was the second-most prevalent gene in patients and healthy carriers, and it had detection rates of 5.1% and 8.1%, respectively. In contrast, afaB was not detected in animal feces or foods, except for three porcine fecal samples. The aggR gene was more prevalent in humans than in foods or animal feces. Both Shiga toxin-producing E. coli and atypical enteropathogenic E. coli carried by cattle may be sources for diarrheal diseases in humans. Pigs may be a source for human enterotoxigenic E. coli infections, whereas humans are expected to be the reservoir for diffusely adhering E. coli, enteroaggregative E. coli, and enteroinvasive E. coli.

Whole genome amplification (WGA) is essential for obtaining genome sequences from single bacterial cells because the quantity of template DNA contained in a single cell is very low. Multiple displacement amplification (MDA), using Phi29 DNA polymerase and random primers, is the most widely used method for single-cell WGA. However, single-cell MDA usually results in uneven genome coverage because of amplification bias, background amplification of contaminating DNA, and formation of chimeras by linking of non-contiguous chromosomal regions. Here, we present a novel MDA method, termed droplet MDA, that minimizes amplification bias and amplification of contaminants by using picoliter-sized droplets for compartmentalized WGA reactions. Extracted DNA fragments from a lysed cell in MDA mixture are divided into 105 droplets (67 pL) within minutes via flow through simple microfluidic channels. Compartmentalized genome fragments can be individually amplified in these droplets without the risk of encounter with reagent-borne or environmental contaminants. Following quality assessment of WGA products from single Escherichia coli cells, we showed that droplet MDA minimized unexpected amplification and improved the percentage of genome recovery from 59% to 89%. Our results demonstrate that microfluidic-generated droplets show potential as an efficient tool for effective amplification of low-input DNA for single-cell genomics and greatly reduce the cost and labor investment required for determination of nearly complete genome sequences of uncultured bacteria from environmental samples.

Polymerase chain reaction assays and culture were used to investigate 728 faecal samples from 404 calves (286 diarrhoeic, 118 healthy) and 324 lambs (230 diarrhoeic, 94 healthy) in Kashmir, India, for the presence of enterotoxigenic Escherichia coli (ETEC), enteroaggregative E. coli (EAEC), diffusely adherent E. coli (DAEC) and salmonellae. Antimicrobial sensitivity patterns were also investigated. In total, 23 ETEC isolates were obtained from the diarrhoeic calves and 12 from diarrhoeic lambs. Most (74%) of the isolates from calves harboured the gene encoding heat-labile enterotoxin I, whereas 75% of the isolates from lambs possessed only the gene encoding for heat-stable enterotoxin a. The ETEC isolates belonged to 20 serogroups, among which serogroups O15 (five isolates) and O8 (four isolates) were the most frequent. Salmonella Typhimurium or S. Enteritidis was identified in three samples from diarrhoeic lambs. The ETEC isolates and the salmonellae showed multidrug resistance. No EAEC or DAEC was detected in any of the samples.

The pathogenesis of diffusely adherent Escherichia coli (DAEC) remains to be elucidated. Previously, we found that afimbrial adhesin gene (afa)-positive motile DAEC strains isolated from patients with diarrhoea induce high levels of IL-8 secretion in Caco-2 cells via toll-like receptor 5 (TLR-5), while non-motile strains did not. The aim of this study was to compare virulence properties, including the phylogenetic groups, afa subtypes, IL-8 secretion levels, and the effects on tight junctions, of DAEC strains isolated from healthy persons with those isolated from patients with diarrhoea. Induction of IL-8 secretion in Caco-2 cells was examined for a total of 36 afa-positive strains: 19 from diarrhoeal patients and 17 from healthy carriers. Irrespective of the source, all strains were classified into the phylogenetic group B2 or D, with the exception of two strains. All 7 motile strains isolated from diarrhoeal patients induced high levels of IL-8 secretion, while only 6 of 15 motile strains from healthy carriers induced IL-8 secretion to the same levels as the diarrhoeal strains. We speculated that additional virulence factors other than afa and motility cause the loosening of tight junctions that allows flagellin to reach TLR-5 located on the basolateral side of the epithelium. However, no differences in the TER and dextran permeability were observed between cells infected with diarrhoeal strains and those from healthy persons. Thus, diarrhoeagenic DAEC seems to possess additional factors, in addition to adhesin and flagellin, which can induce high IL-8 secretion., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Tight junctions contribute to the formation and establishment of intestinal epithelial barriers against microbial infections. However, a variety of enteric pathogens have developed strategies to adhere to and invade epithelial cells and disrupt epithelial integrity. The aim of this study was to ascertain if enterotoxigenic Escherichia coli heat-stable enterotoxin (STa) can cause deterioration of epithelial barrier integrity. Since STa shows amino acid similarity to guanylin, we evaluated the effects of both of these molecules on T84 epithelial cells. T84 epithelial monolayers were grown on 24-well Transwell filters and barrier integrity was assayed by measurement of transepithelial electrical resistance (TER). Macromolecular permeability of the monolayers was determined by measuring the paracellular passage of FITC-labeled dextran 4000 from apical to basolateral compartments of the Transwell filter culture. Treatment of T84 monolayers with either ST or guanylin did not increase paracellular permeability to FITC-dextran. However, although guanylin, which is a physiological guanylate cyclase activator, did not change TER in polarized T84 monolayers, ST did elicit a reduction in TER within 2h, at concentrations above 4μM. These data suggest that STa causes not only induction of water secretion but also intestinal barrier dysfunction., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Aims: The present study aimed to develop a colony hybridization method for the exhaustive detection and isolation of diarrhoeagenic Escherichia coli (DEC) from samples containing numerous coliform bacteria., Methods and Results: Digoxigenin-labelled DNA probes were designed to detect seven pathotypes of DEC based on type-specific genes. A total of 615 meat, food and faeces samples identified as DEC-positive by multiple real-time PCR for the virulence genes (eae, stx, elt, est, virB, aggR, afaB and astA) were analysed by a colony hybridization method, which involved filtering enrichment cultures through hydrophobic grid-membrane filters. DEC were isolated from 72.5% (446/615) of samples by the colony hybridization method but were only detected in 26.3% (162/615) of samples by a conventional culture method. The hybridization method was particularly effective for isolating low-level contaminants, such as enterotoxigenic and Shiga toxin-producing E. coli, which were isolated from 51.8% (58/112) of samples identified as positive by PCR for the enterotoxin genes, in contrast to only 4.5% (5/112) of samples analysed by the conventional method., Conclusions: The developed colony hybridization system allows for the efficient and simultaneous isolation of all DEC pathotypes., Significance and Impact of the Study: The colony hybridization system described here permits the sensitive isolation of DEC and represents a suitable tool for ecological investigations of DEC., (© 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.)

DAEC is considered potentially diarrheagenic. For diffuse adhesion, the role of the Afa, which was originally identified as a uropathogenic factor, is now understood. However, the role of DAEC in diarrheal disease remains controversial because DAEC is often isolated not only from patients but also from healthy individuals. Previously, we suggested that Afa/Dr DAEC, which can induce high levels of IL-8 secretion in cultures of human carcinoma epithelial cells (HEp-2, Caco-2), is enterovirulent. In the present study, we examined whether IL-8 secretion induced by certain Afa/Dr DAEC strains was primarily due to flagella via TLR5. All IL-8 high-inducing strains were highly motile in swarming tests. Partially purified flagella induced IL-8 in a dose-dependent manner. However, IL-8 induction was inhibited by small-interfering RNA against TLR5 or by treating flagella with disialoganglioside-GD1a, a TLR5 blocker. TLR5 is reportedly located on the basolateral side of intestinal epithelia; flagella should not have reached TLR5 from the apical side beyond tight junctions. Reduction in the number of intracellular organisms by wortmannin, a PI3K inhibitor, did not reduce IL-8 secretion. Afa/Dr DAEC seemed to loosen the tight junctions because it quickly reduced transepithelial electrical resistance after infection. Decreased resistance led to increased IL-8 production. In conclusion, diffuse adhesion itself is insufficient to induce high levels of IL-8, and simultaneous stimulation by flagella via TLR5 is likely required for additional induction. Clinically, high motility may be a candidate criterion for predicting the ability of Afa/Dr DAEC strains to induce higher levels of IL-8 secretion.

The etiological roles of diarrheagenic Escherichia coli (DEC), including enteroaggregative E. coli (EAggEC), diffusely adherent E. coli (DAEC) and EAST1EC--a strain of E. coli that possesses no diarrheagenic characteristics other than the EAggEC heat-stable toxin 1 (EAST1) gene--remain controversial. To clarify the prevalence of DEC among healthy individuals in Osaka City, Japan, and to compare the virulence properties of strains previously isolated from diarrheal patients, fecal specimens were examined for DEC. Isolation rates of Shiga toxin-producing E. coli, enterotoxigenic E. coli, and EAggEC were significantly lower among healthy adults than sporadic adult patients. There were no differences in enteropathogenic E. coli (EPEC), DAEC and EAST1EC between patients and healthy carriers. Subtyping of the intimin gene (eae) of EPEC, and measuring the IL-8 inductivity of DAEC on epithelial cells could provide criteria to distinguish strains in diarrheal patients from those in healthy carriers. Proper criteria should be established in order to diagnose subtypes of DEC as causative agents.

Aims: The source and routes of diarrhoeagenic Escherichia coli (DEC) have not been clarified because it is difficult to detect these organisms in samples with numerous coliform bacteria. We have developed multiplex real-time PCR assays for exhaustive detection of DEC., Methods and Results: Primers and TaqMan probes were designed to amplify and quantify one gene (eae, stx1, stx2, elt, est, virB, aggR, astA, and afaB) from each of seven pathotypes of DEC, in duplex or triplex reactions under the same PCR cycling conditions. Specificity was confirmed using 860 strains including 88 DEC strains. The fluorescence threshold cycle and DNA concentrations correlated with decision coefficients of more than 0.99. Subsequently, meat samples and enrichment broths were spiked with DEC and the assays used to detect the genes. The detection limits varied from 7.1 x 10(2) to 1.1 x 10(4) CFU ml(-1), depending on the target genes. All meat samples spiked with a variety of DEC (more than 10 CFU 10 g(-1)) were found to be positive by the method., Conclusions: The present system allows for the efficient and simultaneous determination of various DEC pathotypes., Significance and Impact of the Study: This system makes epidemiological investigations for DEC sensitive and quick, and is a useful tool to clarify the source and routes of DEC.

The role of diffusely adherent Escherichia coli (DAEC) in diarrheal disease has been controversial. However, DAEC strains were recently implicated in diarrheal disease in developing countries. To clarify whether DAEC are prevalent among sporadic cases of diarrheal illness in Osaka City, Japan, E. coli strains isolated between July 1997 and March 2000 during diarrheagenic E. coli (DEC) investigation were retrospectively examined. DAEC strains were recognized among 41 (4.4%) of 924 patients and formed the biggest subgroup of DEC. Previously, we reported that some DAEC strains caused epithelial cells to secrete as much IL-8 as enteroaggregative E. coli strains did. In this study, we attempted to evaluate epidemiologically whether the ability of DAEC to induce IL-8 was involved in the pathogenesis. Relationship among patient age, symptoms, Afa adhesins, season and IL-8 induction were examined. The subgroup of DAEC that possessed Afa genes and/or induced a high level of IL-8 was significantly prevalent among patients age 1 to 4 years; however total DAEC was not significantly high among the children compared to other age group. IL-8 inducing DAEC seems to play a role in causing sporadic diarrheal illnesses, particularly in pediatric fields. Investigations highlighting the relationship between IL-8 induction and enteropathogenicity are clearly necessary to confirm the role of DAEC in infectious enteritis.

Enterohaemorrhagic Escherichia coli O157 (O157) is infectious to humans, particularly children, at very low doses and causes not only haemorrhagic colitis but also other serious symptoms. To investigate an association between intestinal bacterial flora and resistance to such infections, we screened faecal samples for the presence of enteric bacteria that are able to suppress the growth of O157. Samples from 303 individuals, 35 children (aged < or =6 years) and 268 adults (aged 20-59 years), were examined. Colonies with different appearances on sorbitol MacConkey agar medium were screened for the production of bacteriocins inhibitory for O157 in an overlay agar plate assay. O157-inhibiting strains were isolated from 52 individuals. The prevalence of these bacteria tended to rise with age, and was significantly higher among 40- to 59-year-old adults (23/101, 22.8%) than among children (3/35, 8.6%; P<0.05). To test the hypothesis that these bacteriocin-producing strains contribute to resistance against O157 in human adults, we examined faecal samples of 25 healthy O157 carriers. Inhibitory bacteria were more prevalent among the latter (9/25, 36.0%) than among age-matched subjects who did not carry O157 (49/268, 18.3%). It appears, therefore, that inhibitory bacteria in the human gut may play a role in inhibiting propagation of O157 and/or suppressing expression of virulence factors by this pathogen.

Three hundred and twenty-six Escherichia coli isolates recovered from 326 human faecal specimens from sporadic cases of diarrhoea in Kashmir valley, India, were investigated for the presence of stx(1), stx(2), eaeA, hlyA and lt virulence genes. None of the samples was positive for stx genes or Shiga toxins by PCR or enzyme-linked immunosorbent assay. Twenty-three E. coli isolates showed the presence of the eaeA gene, whereas three isolates harboured the lt gene. Enteropathogenic E. coli (EPEC) belonged to 10 different serogroups. Out of 23 EPEC isolates, the majority (78.26%) were atypical while five (21.73%) were typical. Only one of the typical EPEC harboured the EAF plasmid. Subtyping of the eaeA gene showed the presence of eaeA-alpha(1), eaeA-beta, eaeA-xi and eaeA-eta in one, two, four and two isolates, respectively. None of the E. coli isolates possessed eaeA-delta, eaeA-epsilon and eaeA-zeta. This study further upholds the opinion that Shiga toxin-producing E. coli do not pose a major threat to human health in India and eaeA-alpha(1), eaeA-beta, eaeA-xi and eaeA-eta could be common EPEC subtypes prevalent in humans with diarrhoea in India. The present study appears to be the first report of subtype analysis of the eaeA gene from India and also records the isolation of EPEC with the eaeA-xi gene from humans.

Escherichia coli that sparsely adhere to human epithelial cells are known as diffusely adherent E. coli (DAEC), and the role of the Afa/Dr family of adhesins is now understood. Strains that do not possess Afa/Dr, however, comprise another group of DAEC, of which the pathogenicity remains unknown. The ability to induce interleukin-8 (IL-8) secretion from intestinal epithelial cells might be a feature of enterovirulent bacteria. We previously found that some Afa/Dr DAEC strains induce IL-8 by stimulating epithelial cells with flagella. The present study examines whether non-Afa/Dr DAEC can induce IL-8 in epithelial cells (HEp-2, INT407, and T84). Among 21 strains, 11 (52%; 11/21) induced as much IL-8 as high inducer strains of Afa/Dr DAEC. Adhesion did not significantly differ between high and low inducers; therefore diffuse adhesion alone is probably insufficient to induce IL-8. It was shown that IL-8 induction and the number of intracellular bacteria directly correlated. Wortmannin, an inhibitor of the phosphatidylinositol-3-phosphate kinase, reduced both intracellular bacteria and IL-8 secretion. Motile strains were significantly more prevalent among high (10/11) than low (4/10) inducers. However, 4 low invasive strains hardly induced IL-8 despite their motility. In conclusion, some non-Afa/Dr DAEC invoke the induction of high levels of inflammatory cytokines. Unlike Afa/Dr DAEC, however, non-Afa/Dr strains may require invasion to cause strong induction. These non-Afa/Dr high inducers can be enteropathogenic for the cytokine-inducing properties.

Aims: To describe the occurrence and virulence gene pattern of shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) in healthy goats of Jammu and Kashmir, India., Methods and Results: A total of 220 E. coli strains belonging to 60 different 'O' serogroups was isolated from 206 local (nonmigratory) and 69 migratory goats. All the 220 strains were screened for the presence of stx(1), stx(2), eaeA and hlyA genes. Twenty-eight E. coli (75.6%) strains from local and nine (24.3%) strains from migratory goats belonging to 18 different serogroups showed at least presence of one virulence gene studied. Twenty-eight strains (16.47%) (belonging to 13 different serogroups) from local goats carried stx(1) gene alone or in combination with stx(2) gene, while as only one strain (2%) from migratory goats possessed stx(2) gene alone. Interestingly in the present study none of the STEC strains carried eaeA gene. Similarly, none of the strains from local goats possessed eaeA and none of the migratory goats possessed stx(1) gene. Eight strains (16%) (belonging to four different serogroups) from migratory goats carried eaeA gene. Twenty-five (14.7%) and seven (14%) strains from local and migratory goats harboured hlyA gene respectively., Conclusions: Healthy goats of Jammu and Kashmir state serve as a reservoir of STEC and EPEC. Further studies in this direction are needed to work out whether or not they are transmitted to humans in this part of world., Significance and Impact of the Study: This study is the first report of isolation of STEC and EPEC strains from healthy goats in Jammu and Kashmir State of India, which could be a source of infection to humans.

Escherichia coli that adhere sparsely to human epithelial (HEp-2) cells are known as diffusely adherent E. coli(DAEC) and considered potentially diarrheagenic. The role of the afimbrial adhesive sheath (Afa)-identified originally as a uropathogenic factor-in diffuse adhesion is now understood. However, the role of DAEC in diarrheal disease remains controversial. Recently, ability to induce interleukin-8 (IL-8) secretion from intestinal epithelial cells has been suggested as one of the properties of enterovirulent bacteria. In this study, we examined whether DAEC strains possessing Afa genes induced IL-8 in cultures of human carcinoma epithelial cells (e.g., HEp-2, Caco-2, and T84). Nineteen afa-positive DAEC strains were examined for their ability to induce IL-8 secretion, and only 7 strains (37%; 7/19) induced IL-8 as much as enteroaggregative E. coli did. No marked differences in adhesion were observed between high and low inducers. Diffusive adhesiveness itself is unlikely to be sufficient to induce IL-8. All high inducers were motile and others were nonmotile. Additional stimulation by flagella may be required to cause high levels of chemokine induction. Motility or presence of flagella can be an important criterion to predict DAEC diarrheagenicity at clinical laboratories.

Enteroaggregative Escherichia coli heat-stable enterotoxin 1 (EAST1) was originally regarded as a putative enterotoxin of enteroaggregative Escherichia coli. Although its etiological role has not yet been elucidated, it has been epidemiologically suggested that some strains of E. coli possessing EAST1-coding gene (astA) but no other identifiable pathogenic properties comprise a new group of diarrhea-associated E. coli (EAST1EC). However, the source of the organisms and their prevalence in foods are still obscure. In this study, methods for detection of the organisms in foods heavily contaminated with coliforms were evaluated and properties of the isolated strains were compared with those of fecal strains. Four enrichment methods (brilliant green lactose bile broth, E. coli, lauryl tryptose broth, and a combination of brain heart infusion broth and tryptone phosphate broth) were evaluated through inspection of 115 samples. PCR showed positive results in 26 samples after enrichment with a combination of brain heart infusion broth and tryptone phosphate broth, and EAST1EC was successfully isolated from 18 samples. Fifteen samples showed a positive reaction in the PCR test after enrichment by the other methods, and the organisms were isolated from only 10 specimens. The highest prevalence of EAST1EC was found in animal products (16 of 54, 29.6%); the organism was rarely found in foods of plant origin (2 of 45, 4.4%) or fishery products (1 of 16, 6.3%). Although EAST1EC is unexpectedly common in animal products, its potential as a human pathogen remains uncertain because the possession of some virulence properties differs significantly between strains from fecal specimens and those from foods. Some food isolates, however, possess the same characteristics as diarrheal isolates do. It is necessary to clarify the pathogenicity of EAST1EC and the significance of food as a source of infection.

Aims: To investigate the presence or absence of shiga toxin-producing Escherichia coli (STEC) in avian species in India., Methods and Results: Faecal samples originating from 500 chicken and 25 free flying pigeons were screened for the presence of E. coli. A total of 426 (chicken, 401; pigeons, 25) E. coli strains were isolated. Of 426 E. coli strains, 387 were grouped into 77 serogroups, while 70 and 59 strains were untypable and rough, respectively. All isolates were subjected to multiplex polymerase chain reaction (m-PCR) for the detection of stx(1), stx(2), eaeA, hlyA and saa genes. None of the E. coli strains studied showed the presence of stx(1), stx(2) or their variants and saa genes. Overall 11 (2.74%) and seven (1.74%) strains from chickens possessed eaeA and hlyA genes, respectively, while as only six (1.49%) strains from chickens possessed both eaeA and hlyA genes. O9, O8, O60 and O25 serogroups were most predominant of which there were 24 (5.63%), 23 (5.39%), 23 (5.39%) and 20 (4.69%) strains, respectively. None of the isolates from pigeons showed the presence of any of the virulence genes studied., Conclusions: STEC are absent in chickens and pigeons. However, further studies are required in this direction to confirm or contradict our findings. E. coli strains originating from birds are carrying a low percentage eaeA or hlyA genes., Significance and Impact of the Study: The present study is the first attempt to investigate STEC in chickens and free flying pigeons in India. The chickens and pigeons cannot be considered as important carrier of STEC in India.

Aims: To determine the prevalence and molecular characteristics of Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) in calves and lambs with diarrhoea in India., Methods and Results: Faecal samples originating from 391 calves and 101 lambs which had diarrhoea were screened for presence of E. coli. A total number of 309 (249 bovine and 60 ovine) E. coli strains were isolated. A total of 113 bovine and 15 ovine strains were subjected to multiplex polymerase chain reaction (m-PCR) for detection of stx1, stx2, eaeA and EHEC hlyA genes. STEC and EPEC belonging to different serogpoups were detected in 9.73% of calves studied. Six per cent and 26.66% of lambs studied were carrying STEC and EPEC, respectively. Majority of the STEC serogroups isolated in this study did not belong to those which have been identified earlier to be associated mainly with diarrhoea and enteritis in cattle and sheep outside India. The most frequent serogroup among bovine and ovine EPEC was O26 (40%). One of the most important STEC serogroup O157, known for certain life-threatening infections in humans, was isolated from both bovine and ovine faecal samples., Conclusions: A high percentage of STEC and EPEC belonging to different serogroups are prevalent in calves and lambs with diarrhoea in India and could be the cause of disease in them., Significance and Impact of the Study: The study reports, for the first time, the isolation and characterization of STEC and EPEC serogroups associated with diarrhoea in calves and lambs in India. Many STEC and EPEC strains belonged to serogoups known for certain life-threatening diseases in humans.

Diarrheagenic Escherichia coli (DEC) represents an elusive target, since they are not easily distinguished from fecal coliforms. To clarify if DEC are prevalent among sporadic cases of diarrheal illness in Osaka City, Japan, diarrheal specimens were examined for E. coli that were enterohemorrhagic (EHEC), enteropathogenic (EPEC), enterotoxigenic (ETEC), enteroinvasive (EIEC), or enteroaggregative (EAggEC). EAST1EC, a strain of E. coli that does not possess any diarrheagenic characteristics except the EAggEC heat-stable toxin 1 (EAST1) gene, was also included as a possible DEC. A total of 924 specimens were examined between July 1997 and March 2000. DEC and Salmonella were isolated from 7.3% (67/924) and 6.8% (63/924) of specimens, respectively. DEC was therefore as prevalent as Salmonella among sporadic cases. The 67 strains were composed of 17 EPEC (26%), 10 EHEC (15%), four ETEC (6%), 13 EAggEC (20%), and 23 EAST1EC (35%), including two strains of EAST1EC O166:H15. Although PCR and tissue culture adhesion tests were useful to detect DEC, the effectiveness of serotyping was limited: only 40 strains (17.5%) out of 229 isolates that had been assumed to be enterovirulent on the basis of their O antigen were recognized to be diarrheagenic. In conclusion, not only EHEC but also the other subgroups of DEC, including EAST1EC, seem to play an important role in causing sporadic diarrheal illnesses. Methods to detect and unified criteria to identify various kinds of DEC are strongly desired.

In an outbreak of gastroenteritis on 23 July 1996, in Osaka, Japan, 54 of 91 persons who had attended a meeting the previous day became ill. Escherichia coli O166:H15 was isolated from stool specimens of patients (29/33, 88%). Laboratory tests for other bacterial pathogens and viruses were negative. The E. coli 0166 organisms did not adhere to HEp-2 cells in a localized, diffuse, or enteroaggregative manner. The organisms did not express known enterotoxigenic E. coli (ETEC) colonization factors. In polymerase chain reaction tests, the bacteria did not have coding genes for shigatoxin of enterohemorrhagic E. coli (EHEC), heat-labile, or heat-stable enterotoxin of ETEC, attachment and effacement (eaeA) of EPEC, or invasion (invE) of enteroinvasive E. coli (EIEC). Consequently, they could not be assigned to any of the recognized diarrhoeagenic groups of E. coli: EPEC, ETEC, EHEC, EIEC, enteroaggregative E. coli (EAggEC), or diffusely adhering E. coli. However, the organisms possessed the EAggEC heat-stable enterotoxin (EAST1) gene. To our knowledge, this is the first report of an outbreak caused by E. coli that did not have well-characterized virulence genes other than EAST1. The isolates showed the same DNA banding pattern in pulsed-field gel electrophoresis after digestion with the restriction enzymes XbaI or NotI. Three O166:H15 strains isolated from two sporadic cases and another outbreak during 1997-8 were distinct, indicating that multiple clones have spread already. We propose that diarrhoeal specimens should be examined for E. coli possessing the EAST1 gene.

Enterotoxigenic Escherichia coli (ETEC) serotype O169:H41 organisms have become the most prevalent ETEC in Japan since the first outbreak in 1991. It was assumed that the outbreaks were due to clonal spread of this new ETEC serotype. The relationship of 32 strains isolated from 6 outbreaks were examined for biotype, antibiotic susceptibility, enterotoxigenicity, protein banding pattern, lipopolysaccharide banding pattern, plasmid analysis, and ribotyping. Further, the strains were examined by haemagglutination, surface hydrophobicity, and the ability to adhere to HEp-2 cells. The present study suggests that the outbreaks were caused by multiple clones of STp-producing O169:H41 since they showed differences in ribotype and outer membrane protein banding patterns. The strains did not agglutinate human or bovine red blood cells in a mannose-resistant manner. They adhered to HEp-2 cells in a manner resembling enteroaggregative E. coli. Five strains were examined by dot-blot tests for the colonization factor antigens CFA/I, CS1, CS2, CS3, CS4, CS5, CS6, CS7, PCFO159, PCFO166 and CFA/III. Although four strains expressed CS6, no structure for CS6 was identified. A strain that the anti-CS6 MAbs did not react with could adhere to HEp-2 cells in mannose resistant manner; thus, it is unlikely that CS6 play an important role in the adhesion to the cells. Electron microscopy studies of the O169:H41 strains suggested that curly fimbriae, a possible new colonization factor, may be playing an important role in the adhesion of the bacteria to HEp-2 cells. In conclusion, outbreaks due to ETEC O169:H41 were caused by multiple clones, and the strains should be examined in detail for a possible new colonization factor.

The virulence traits that mediate Vero cytotoxin-producing E. coli (VTEC) adherence are unclear. Many VTEC strains possess the eaeA gene which is involved in the attaching and effacing effects of enteropathogenic E. coli (EPEC). Most eae-positive VTEC adhered to HEp-2 cells in a localized manner; however some strains did not adhere. Thus we investigated the adhesion of poorly adherent strains, especially those of serogroups O111 and O157. To establish a model, the adherence to HEp-2, INT407 and Caco-2 cells of 12 O157 VTEC and six O111 VTEC isolated from cases of human infection were studied after growth of the bacteria under different conditions. For adhesion tests mannose is usually added during prior broth culture of the bacteria, and during the period of attachment, so that any adhesion due to mannose-sensitive type 1 pili is inhibited. Bacteria cultured in peptone water in the absence of mannose adhered to all three lines; there were localized clusters of bacteria on 1%-82% cells, whether mannose was present during the attachment period or not. Bacteria grown in the presence of D-mannose, or any other sugar that was metabolized, showed little adherence (range 0-9%). alpha-Methyl-glucoside also caused marked inhibition of adhesion. It was concluded that inhibition of adhesion was due to catabolite repression.

Mutants of Escherichia coli tolerant to the ghosts of T-even phages (T2, T4, and T6) have been isolated from a strain supersensitive to T6 phage. First, T6 supersensitive mutants were isolated from mutagenized E. coli W2252 by replica plating to T6 phage-overlaid agar. One of them, strain NM101, was mutagenized again, grown, and then plated with a high multiplicity of T4 and T6 ghosts. Surviving cells were checked for tolerance to ghosts and adsorption of phages. One such ghost-tolerant mutant, strain GT29, was tolerant to ghosts of both T4 and T6 phages and sensitive to T2 ghosts. This mutant was also sensitive to ethylenediaminetetraacetic acid and penicillin G and intermediately sensitive to acriflavine, sodium dodecyl sulfate, sodium deoxycholate, actinomycin D, and lysozyme. Another mutant, strain GT62, was tolerant not only to T4 and T6 ghosts but also to T2 ghosts. It was sensitive to sodium dodecyl sulfate, sodium deoxycholate, penicillin G, acridine orange, actinomycin D, phenethyl alcohol, and novobiocin and intermediately sensitive to acriflavine and lysozyme. Spontaneous revertants of strain GT62 were isolated with a frequency of 2.7 X 10(-9). It is suggested that ghosts attack host bacteria indirectly through the cell surface by a mechanism similar to the transmission hypothesis that was originally adopted by Nomura (1967) to explain the mechanism of the action of colicins, and that our ghost-tolerant mutants presumably have defects in the cell surface.

The influence of ovarian hormones on the adhesion of Escherichia coli to endometrial epithelial cells was investigated in an in vitro system. Endometrial cells liberated by collagenase from rat uteri were used. Optimal test conditions were obtained when 5 X 10(8) E. coli bacteria were added to 10(5) epithelial cells and incubated for 60 min. The adhesion of the organisms was inhibited by the addition of either mannose or alpha-methyl-D-mannopyranoside. When epithelial cells collected from uteri of estradiol-treated rats were used, the number of E. coli adhering to the cells was markedly lower than that adhering to epithelial cells collected from control rats. These results suggest that E. coli adheres to endometrial epithelial cells with so-called type 1 pili and that estradiol alters the nature of the endometrial epithelium and prevents the adherence of the organisms to the cells.

Escherichia coli was inoculated into the uterine lumen of ovariectomized rats, and the endometrial surfaces were examined by scanning electron microscopy. Adherence of E. coli to the epithelium and destruction of the surface leading to purulent endometritis were noticed. When rats were treated previously with estradiol, adherence of E. coli was not detected.

Aim This study assessed whether multilocus variable-number tandem repeat analysis ( MLVA) and antimicrobial susceptibility testing discriminated diarrhoeagenic atypical enteropathogenic Escherichia coli ( aEPEC) from aEPEC indigenous to domestic animals or healthy people. Methods and Results MLVA genotyping of 142 aEPEC strains isolated from foods and faecal samples of domestic animals and humans revealed 126 distinct MLVA profiles that distributed to four clusters, yielding a Simpson's index of diversity ( D) of 99·8%. Cluster 2 included 87% of cattle isolates and 67% of patient isolates. The plurality (15/34, 44%) of strains from healthy humans mapped to Cluster 1, while half (18/41, 44%) of the swine strains belonged to Cluster 4. Testing for antimicrobial susceptibility revealed that 52 strains (37%) of aEPEC were resistant to one or more agents; only 10 strains (7%) exhibited resistance to more than three agents. Strains isolated from swine or food exhibited a wider variety of resistance phenotypes than bovine or human strains. Conclusions MLVA assigned the aEPEC isolates from cattle and patients to Cluster 2, distinct from aEPEC from other sources. Hog yards may be a larger source of drug-resistant strains than are cattle ranches. Significance and Impact of the Study MLVA suggests that human diarrhoeagenic aEPEC are derived from cattle and are distinct from strains carried by healthy people and other animals. Cattle appear to be reservoirs of human diarrhoeagenic aEPEC. [ABSTRACT FROM AUTHOR]

Shigatoxigenic Escherichia coli (STEC) are major food-borne pathogens. They transmit to human through contaminated meat and meat products of animals and poultry, and frequently associated with various types of human illness including haemolytic uremic syndrome. This preliminary study showed the prevalence of STEC in 60 cloacal swab samples of live healthy broiler chickens collected randomly when sold at a wholesale market in Mymensingh district of Bangladesh. Isolation and identification of E. coli was carried out using Eosin Methylene Blue (EMB) agar media and 16S rRNA gene specific polymerase chain reaction (PCR). Among the 60 samples, 49 (81.67%) were found positive to E. coli. These E. coli isolates were screened for the detection of STEC by PCR using stx1 and stx2 gene specific primers. Among the 49 positive samples, 5 (10.20%) were found positive for stx1 gene, and 26 (53.06%) were positive for stx2 gene. In addition, 6 (12.24%) isolates were found positive to both stx1 and stx2 genes, and the remaining 12 (22.46%) were negative. The high prevalence of STEC in the broiler chicken alarms the public health impact as the people are always in close contact with these live broiler chickens in the open market as well as processing of meat at home before cooking. However, further studies are required to uncover the major source(s) for the transmission of STEC to human in rural Bangladesh. [ABSTRACT FROM AUTHOR]

Fifty-eight typical EAEC isolates from children with diarrhoea were examined for HEp-2 cell adherence assay, presence of dispersin ( aap), yersiniabactin ( irp2), plasmid encoded toxins ( pet), Shigella enterotoxin1 ( set1A) and cryptic open reading frame ( shf) putative virulence genes by polymerase chain reaction as well as for biofilm production. All the isolates showed aggregative adherence pattern on HEp-2 cells. All but five isolates (91.3 %) carried aap gene. While irp2, pet, set1A and shf genes were detected in 68.9, 5.1, 39.6, and 60.3 % isolates, respectively. Thirty-three (64.7 %) isolates out of 51 tested were found to produce biofilm which was found to be significantly associated only with set1A virulence gene ( P = 0.025). Highest amount of biofilm was produced by a strain that possessed all the genes studied. Out of 14 isolates in which the most frequent gene combination ( aap, irp2 and shf) was observed, only six produced biofilm. It is concluded that there is significant heterogeneity in putative virulence genes of EAEC isolates from diarrhoeic children and biofilm formation is associated with multiple genes. [ABSTRACT FROM AUTHOR]

Seven hundred and thirty-five diarrhoeic faecal samples from children were investigated for presence of enteroaggregative E. coli (EAEC), enterotoxigenic E. coli (ETEC), diffusely adherent E. coli (DAEC) and Salmonella spp. by polymerase chain reaction (PCR) and bacterial culture. Out of 675 samples from Kashmir, 55 isolates were obtained, which carried at least one virulence gene studied. Out of the 55 isolates, 36 (65.45%) were EAEC, 18 (32.72%) were ETEC while only one isolate (1.81%) was DAEC. All the EAEC isolates were found to be typical as they possessed aggR gene. Six (16.66%) EAEC isolates carried the astA gene. Out of the 18 ETEC isolates, 13 carried the elt gene alone, four possessed both the elt and est genes and the remaining one harboured the est gene alone. Five ETEC isolates also possessed astA gene. Nineteen EAEC isolates belonged to 10 different serogroups. Serogroup O153 was most frequent. The ETEC isolates also belonged to 10 different serogroups of which O159 was most predominant. Out of 224 E. coli isolates from 60 samples of Secunderabad, 27 isolates carried at least one virulence gene. Out of 27 isolates 22 (81.48%) were typical EAEC, three (11.11%) were ETEC and two (7.4%) were DAEC. Fifteen EAEC isolates belonged to seven different serogroups with O86 as most frequent. Four EAEC isolates also possessed the astA gene. All the three ETEC isolates harboured elt gene only and belonged to three different serogroups. Two isolates of Salmonella Worthington were obtained from only two samples in Kashmir. [ABSTRACT FROM AUTHOR]

Abstract: This communication describes the prevalence and virulence attributes of Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) isolates in lambs with and without diarrhoea in Kashmir, India. One hundred twenty and 164 E. coli isolates belonging to 56 different ‘O’ serogroups were isolated from 101 diarrhoeic and 135 healthy lambs, respectively. All the 284 isolates were screened for presence of stx 1 , stx 2 , eae and ehxA genes using multiplex polymerase chain reaction (m-PCR). Forty four (36.67%) isolates from lambs with diarrhoea carried at least one virulence gene studied. Twenty one (17.5%) and 15 (12.5%) isolates from diarrhoeic lambs were STEC and EPEC, respectively. Thirty (18.3%) isolates from healthy lambs possessed at least one virulence gene studied. Fourteen (8.53%) and 16 (9.75%) isolates from healthy lambs were detected as STEC and EPEC, respectively. Sixteen (45.71%) of 35 STEC isolates from both diarrhoeic and healthy lambs, carried both stx 1 and stx 2 genes, 15 (42.85%) had stx 1 alone and four (11.42%) had stx 2 gene alone. Statistically the difference between prevalence of STEC in diarrhoeic and healthy lambs was significant but that of EPEC was insignificant. One (2.85%) and 28 (80%) of STEC isolates possessed eae and ehxA genes, respectively. The high percentage of STEC and EPEC prevalent in diarrhoeic and healthy lambs may serve as source of infection to humans. STEC seem to be associated with diarrhoea in lambs. [Copyright &y& Elsevier]

Aims: To determine the subtypes of stx and eae genes of Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic Escherichia coli (EPEC) from calves and to ascertain the typical and atypical nature of EPEC. Methods and Results: One hundred and eighty-seven faecal samples from 134 diarrhoeic and 53 healthy calves were investigated for the presence of stx, eae and ehxA virulence genes by polymerase chain reaction and enzyme-linked immunosorbent assay. Subtype analysis of stx 1 exhibited stx 1c in 13 (31·70%) isolates, while that of stx 2 revealed stx 2c in eight (24·24%) and stx 2d in two (6·06%) isolates. Subtyping of eae gene showed the presence of eae-β, eae-η and eae-ζ in two, three and four isolates respectively. None of the E. coli isolates possessed stx 2e, stx 2f, eae-α, eae-δ, eae-ε and eae-ξ. All EPEC isolates were atypical. Conclusions: stx 1, stx 1c, stx 2, stx 2c, stx 2d, eae-β, eae-η and eae-ζ subtypes are prevalent in STEC and EPEC isolates in India. Significance and Impact of the Study: This is the first subtype analysis of stx 2 and eae genes of animal E. coli isolates in India and emphasizes the need to investigate their transmission to humans. [ABSTRACT FROM AUTHOR]

s.a. wani, i. samanta, m.a. bhat and y. nishikawa. 2004.To investigate the presence or absence of shiga toxin-producingEscherichia coli(STEC) in avian species in India.Faecal samples originating from 500 chicken and 25 free flying pigeons were screened for the presence ofE. coli. A total of 426 (chicken, 401; pigeons, 25)E. colistrains were isolated. Of 426E. colistrains, 387 were grouped into 77 serogroups, while 70 and 59 strains were untypable and rough, respectively. All isolates were subjected to multiplex polymerase chain reaction (m-PCR) for the detection ofstx1,stx2,eaeA,hlyAandsaagenes. None of theE. colistrains studied showed the presence ofstx1,stx2 or their variants andsaagenes. Overall 11 (2·74%) and seven (1·74%) strains from chickens possessedeaeAandhlyAgenes, respectively, while as only six (1·49%) strains from chickens possessed botheaeAandhlyAgenes. O9, O8, O60 and O25 serogroups were most predominant of which there were 24 (5·63%), 23 (5·39%), 23 (5·39%) and 20 (4·69%) strains, respectively. None of the isolates from pigeons showed the presence of any of the virulence genes studied.STEC are absent in chickens and pigeons. However, further studies are required in this direction to confirm or contradict our findings.E. colistrains originating from birds are carrying a low percentageeaeAorhlyAgenes.The present study is the first attempt to investigate STEC in chickens and free flying pigeons in India. The chickens and pigeons cannot be considered as important carrier of STEC in India. [ABSTRACT FROM AUTHOR]

The present study has determined the serological diversity, virulence-gene profile and in vitro antibiogram of avian pathogenic Escherichia coli (APEC) isolates from broiler chickens in India suspected to have died of colibacillosis. The virulence-gene profile of APEC was compared with that of the Escherichia coli isolates from faeces of apparently healthy chickens, called avian faecal E. coli (AFEC). In total, 90 representative isolates of APEC and 63 isolates of AFEC were investigated in the present study. The APEC were typed into 19 serogroups, while some isolates were rough and could not be typed. Most prevalent serogroup was O2 (24.44%). Among the eight virulence genes studied, the prevalence of seven genes (iss , iucD , tsh , cva / cvi , irp2 , papC and vat) was significantly higher in APEC than in AFEC isolates. However, there was no significant difference between APEC and AFEC isolates for possession of astA gene. The most frequent gene detected among the two groups of organisms was iss , which was present in 98.88% and 44.44% of APEC and AFEC isolates respectively. The in vitro antibiogram showed that the majority (96.6%) of APEC isolates were resistant to tetracycline, while 82.2% were resistant to cephalexin, 78.8% to cotrimoxazole, 68.8% to streptomycin and 63.3% to ampicillin. However, most of them (84.45%) were sensitive to gentamicin. Thus, it is concluded that APEC from the broiler chickens carried putative virulence genes that attributed to their pathogenicity. Furthermore, the majority of APEC isolates were found to be multi-drug resistant, which, in addition to leading treatment failures in poultry, poses a public health threat. Avian colibacillosis caused by avian pathogenic Escherichia coli (APEC) is responsible for heavy economic losses to commercial broiler farmers. The present work investigated APEC isolates and their comparison with avian faecal E. coli (AFEC) isolates. The results suggest that virulence genes were more prevalent in APEC than in AFEC and the majority of the APEC isolates exhibited a multi drug resistant pattern. The present study will serve as a reference study for future work on APEC and extra intestinal pathogenic E. coli. [ABSTRACT FROM AUTHOR]

Seventy-five Escherichia coli isolates with at least one targeted virulence gene were recovered from lambs with (n = 230) and without (n = 108) diarrhoea. The isolates belonged to 36 different serogroups. Shiga toxin-producing E. coli (STEC) was isolated from 9.6% of lambs with and 24.1% of lambs wit diarrhoea. Enteropathogenic E. coil (EPEC) was isolated from 6.1% of lambs with and 11.1% of lambs wit out diarrhoea. Of 26 EPEC isolates, seven were typical (positive for bfpA), and, of 34 stxl positive isolates, 25 were subtyped as stx2. Five of 29 stx2 positive isolates were subtyped as stx2d and two as stx2c. Seven of 45 eae positive isolates were subtyped as eae subtype zeta (eaeζ). This appears to be the first report of the isolation of typical EPEC from sheep in India. [ABSTRACT FROM AUTHOR]