Enteroaggregative Escherichia Coli Research Articles

A large family of antivirulence regulators modulates the effects of transcriptional activators in Gram-negative pathogenic bacteria.

We have reported that transcription of a hypothetical small open reading frame (orf60) in enteroaggregative E. coli (EAEC) strain 042 is impaired after mutation of aggR, which encodes a global virulence activator. We have also reported that the cryptic orf60 locus was linked to protection against EAEC diarrhea in two epidemiologic studies. Here, we report that the orf60 product acts as a negative regulator of aggR itself. The orf60 protein product lacks homology to known repressors, but displays 44–100% similarity to at least fifty previously undescribed small (<10 kDa) hypothetical proteins found in many gram negative pathogen genomes. Expression of orf60 homologs from enterotoxigenic E. coli (ETEC) repressed the expression of the AraC-transcriptional ETEC regulator CfaD/Rns and its regulon in ETEC strain H10407. Complementation in trans of EAEC 042orf60 by orf60 homologs from ETEC and the mouse pathogen Citrobacter rodentium resulted in dramatic suppression of aggR. A C. rodentium orf60 homolog mutant showed increased levels of activator RegA and increased colonization of the adult mouse. We propose the name Aar (AggR-activated regulator) for the clinically and epidemiologically important orf60 product in EAEC, and postulate the existence of a large family of homologs among pathogenic Enterobacteriaceae and Pasteurellaceae. We propose the name ANR (AraC Negative Regulators) for this family.

An investigation of the diversity of strains of enteroaggregative Escherichia coli isolated from cases associated with a large multi-pathogen foodborne outbreak in the UK.

Following a large outbreak of foodborne gastrointestinal (GI) disease, a multiplex PCR approach was used retrospectively to investigate faecal specimens from 88 of the 413 reported cases. Gene targets from a range of bacterial GI pathogens were detected, including Salmonella species, Shigella species and Shiga toxin-producing Escherichia coli, with the majority (75%) of faecal specimens being PCR positive for aggR associated with the Enteroaggregative E. coli (EAEC) group. The 20 isolates of EAEC recovered from the outbreak specimens exhibited a range of serotypes, the most frequent being O104:H4 and O131:H27. None of the EAEC isolates had the Shiga toxin (stx) genes. Multilocus sequence typing and single nucleotide polymorphism analysis of the core genome confirmed the diverse phylogeny of the strains. The analysis also revealed a close phylogenetic relationship between the EAEC O104:H4 strains in this outbreak and the strain of E. coli O104:H4 associated with a large outbreak of haemolytic ureamic syndrome in Germany in 2011. Further analysis of the EAEC plasmids, encoding the key enteroaggregative virulence genes, showed diversity with respect to FIB/FII type, gene content and genomic architecture. Known EAEC virulence genes, such as aggR, aat and aap, were present in all but one of the strains. A variety of fimbrial genes were observed, including genes encoding all five known fimbrial types, AAF/1 to AAF/V. The AAI operon was present in its entirety in 15 of the EAEC strains, absent in three and present, but incomplete, in two isolates. EAEC is known to be a diverse pathotype and this study demonstrates that a high level of diversity in strains recovered from cases associated with a single outbreak. Although the EAEC in this study did not carry the stx genes, this outbreak provides further evidence of the pathogenic potential of the EAEC O104:H4 serotype.

Enteroaggregative Escherichia coli in diarrheic children in Egypt: molecular characterization and antimicrobial susceptibility.

Little information is available regarding the significance of enteroaggregative Escherichia coli (EAEC) in pediatric diarrhea in Egypt. Escherichia coli was isolated from stool samples of 62 diarrheic and 43 non-diarrheic (control) Egyptian children. Samples were screened for genes specific for enteroaggregative E. coli (EAEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), Shiga toxin-producing E. coli (STEC), and enteroinvasive E. coli (EIEC) using polymerase chain reaction (PCR). Diarrheagenic E. coli were grouped phylogenetically using PCR and tested for their susceptibility to antibiotics using the disk diffusion method. Isolates designated as EAEC were examined for eight virulence factors (VFs) using PCR. EAEC was detected in 19 (30.7%) and 4 (9.3%), EPEC in 2 (3.2%) and 1 (2.3%), and ETEC in 2 (3.2%) and 0 (0.0%) diarrheic and control children, respectively; STEC and EIEC were not detected. Only EAEC was significantly isolated from diarrheic children compared with controls (p < 0.01, OR = 4.31).Three or more VFs (multivirulent isolates) were found in 52.6% and 50% of EAEC isolated from diarrheic children and controls, respectively. More than 73% (17/23) of EAEC isolates were identified as belonging to phylogenetic group D. Multiple-antibiotic resistance (resistance to three or more drugs) was observed in more than 91% of EAEC. Multivirulent EAEC is a significant causative agent of pediatric diarrhea in Egypt, with the majority of isolated EAEC belong to phylogenetic group D. Multiple-antibiotic resistance among EAEC has the potential to be a serious public health problem for the country.

English

Molecular identification and antimicrobial susceptibility of enteroaggregative Escherichia coli (EAEC) associated with childhood diarrhea was done out in Egypt. The usefulness of quantitative biofilm assay in detection of EAEC was compared with multiplex polymerase chain reaction (PCR). One hundred and fifty cases of childhood diarrhea were divided into three groups; 50 cases of acute diarrhea (group I), 50 cases of persistent diarrhea (group II) and 50 cases of healthy subjects of matched age and sex as a control group (group III). E. coli was isolated and identified by conventional microbiological methods. EAEC was detected by multiplex PCR and quantitative biofilm assay. Antimicrobial susceptibility profile of the isolated EAEC strains was done using disc diffusion method. E. coli was isolated from 78% (39/50) cases of acute diarrhea and 76% (38/50) cases of chronic diarrhea. The results show no significant difference between the results of multiplex PCR and quantitative biofilm assay; in 77 E. coli isolates, 15 (19.5 %) generated positive results for EAEC with multiplex PCR for two the specific genes AggR and EAST and 12 (15.6 %) strains showed positive results for EAEC by quantitative biofilm assay. As regard the antimicrobial susceptibility profile of the isolated EAEC strains, the results show that 85.7 and 87.5% of the EAEC strains isolated from cases of acute and persistent diarrhea, respectively were sensitive to amikacin, 47.1 and 62.5% were sensitive to cefoperazone, 28.5 and 50.00% were sensitive to ceftriaxone and 42.8% and 62.5% were sensitive to imepenem, 28.5 and 12.5 % of the EAEC strains isolated from cases of acute and persistent diarrhea respectively were sensitive to Amoxicillin-Clavulanic. All the isolated EAEC strains (100.00%) were resistant to sulphamethoxole/trimethoprim. High incidence of EAEC associated diarrhea among pediatric cases in Egypt must be considered before decision of antimicrobial therapy. Quantitative biofilm assay can be simple, rapid and convenient method for detection of EAEC in comparison with molecular methods and can therefore be recommended as a rapid screening test for EAEC in clinical laboratories. &nbsp; Key words: Enteroaggregative Escherichia coli (EAEC), infantile diarrhea, multiplex polymerase chain reaction (PCR), quantitative biofilm.

Enteroaggregative Escherichia Coli (EAEC) in South of Iran

Introduction: The aim of the present study was to investigate the presence and the frequency of EAEC as etiologic agent of diarrhea in Shiraz. Enteroaggregative E. coli (EAEC) is increasingly recognized as a cause of often persistent diarrhoea in children and adults in both developing and developed countries, and have been identified as the cause of several outbreaks worldwide. Materials and Method: A total of 715 stool samples were collected from patients with diarrhea in Shiraz. Diarrheagenic E. coli were isolated by biochemical tests and culture from 715 stool samples collected from different hospitals. Diarrheagenic E. coli strains isolated from diarrheal stool samples were examined for the detection of the aggR gene by Real time PCR and PCR method. Results: In this study, a total of 101 (14.12%) diarrheagenic E. coli were isolated from 715 stool samples collected from different hospitals. Diarrheagenic E. coli were isolated much more frequently in the summer months than other season. Out of these 101 diarrheagenic E. coli identified, 5 were confirmed as EAEC in patient. The high prevalence of EAEC isolates was also found in watery diarrhea. Conclusion: We therefore, recommend the routine isolation and identification of EAEC strains from patient with diarrhea in all the clinical laboratories and other pathotype diarrhoeagenic E. coli in Iran. Keywords: Diarrhea, Enteroaggregative Escherichia coli (EAEC), Real-Time PCR.

Genotypic and Phenotypic Comparison of Enteroaggregative Escherichia coli Isolates from HIV-Positive and Non-HIV Diarrheal Samples

Enteroaggregative Escherichia coli (EAEC) have been isolated from both HIV-positive and non-HIV diarrheal samples. In this study a collection of 18 isolates from these two groups were compared for biofilm formation and antibiotic resistance and for the presence of 14 virulence-related genes. All the HIV-positive and over 66% of the non- HIV strains were PCR-negative for adhesion-related sequences indicating that as yet unknown adhesins may play a role. However, despite some variations, the prevalence rate of the virulence-related genes was not significantly different in the two groups. HIV-positive isolates were biofilm producer but only a single weak biofilm former was observed among the non-HIV strains. The rate of resistance to most of the antibiotics used was higher among the HIV-positive group than the non-HIV isolates, but was significantly higher for amoxicillin-calvulanic acid (100%) and nalidixic acid (55.5%). Pulse field gel electrophoresis of the isolates produced 17 unique profiles reflecting the exiting heterogeneity of the isolates.

Enteroaggregative coli: A Pathogen Bridging the North and South.

Enteroaggregative Escherichia coli (EAEC) is a heterogeneous emerging enteric pathogen. Identified during the 1980's when EAEC strains where isolated from cases of acute and persistent diarrhea among infants from developing countries and of traveler's diarrhea. Subsequently, EAEC strains were linked with foodborne outbreaks and diarrhea illness in adults and children from industrialized countries, HIV-infected subjects and stunting of malnourished poor children. Nowadays, EAEC is increasingly recognized as a major cause of acute diarrhea in children recurring hospitalization and of traveler's diarrhea worldwide. EAEC strains defining phenotype is the aggregative adherence (AA) pattern on epithelial cells. AggR a transcriptional regulator of several EAEC virulence genes has been a key factor in both understanding EAEC pathogenesis and defining typical EAEC (tEAEC) strains. EAEC virulence genes distribution among these strains is highly variable. Present challenges are the identification of key virulence genes and how they coordinately function in the setting of enteric disease.

In silico and in vivo studies of truncated forms of flagellin (FliC) of enteroaggregative Escherichia coli fused to FimH from uropathogenic Escherichia coli as a vaccine candidate against urinary tract infections

The new generation of vaccines against infectious diseases is based on recombinant fusion proteins. Flagellin (FliC) of enteroaggregative Escherichia coli (EAEC) could be considered as a potent adjuvant in designing new vaccines. However, because of its large size, incorporation of this protein with a vaccine antigen might negatively influence recognition of the vaccine epitopes by the immune system. Designing the truncated forms of FliC, capable of inducing innate immune response, enhances the immune responses to the target antigen. We have previously shown that two truncated forms of FliC are able to induce Interleukine-8 production in HT-29 epithelial cell line. In this study we designed recombinant vaccine against urinary tract infections (UTIs) using truncated forms of FliC and type 1 fimbrial FimH adhesin from uropathogenic Escherichia coli (UPEC) and studied their in silico interactions with Toll-like receptor 5 (TLR-5) via docking protocols. The best fusion protein was subjected to cloning and expression. The ability of the recombinant vaccine and the truncated forms in inducing immune responses was investigated. Our results showed that truncated forms are capable of inducing Th1 (forms A and B) and Th2 (form A) responses and fusion vaccine induced strong cellular and humoral immune responses.

Characterization of CTX-M-Type Extended-Spectrum Beta-Lactamase-Producing Diarrheagenic Escherichia coli Isolates in the Republic of Korea During 2008-2011

To characterize the extended-spectrum beta-lactamases (ESBLs) in diarrheagenic Escherichia coli from Korea in 2008-2011, we screened seven enterotoxigenic E. coli (ETEC) and one enteroaggregative E. coli (EAEC) that produce ESBLs from a nationwide survey. All eight isolates produced CTX-M-type ESBLs, including CTX-M-12 (n = 4), CTX-M-14 (n = 2), and CTX-M-15 (n = 2). PCR-based replicon typing indicated that the blaCTX-M-12 genes of four ETEC isolates were carried on a conjugative IncF plasmid, whereas the blaCTX-M-14 of one EAEC was located on an IncK plasmid. This is the first report of the occurrence of blaCTX-M genes in clinical isolates of EAEC in Korea. The ESBL-producing isolates were shown to be different based on pulsed-field gel electrophoresis and multilocus sequence typing, whereas the four isolates with CTX-M-12 were clonally related. These observations raise an alarm for the spread of plasmid-mediated resistance to ESBL among diarrheagenic E. coli.

Identification of Cell Surface-Exposed Proteins Involved in the Fimbria-Mediated Adherence of Enteroaggregative Escherichia coli to Intestinal Cells

Fimbria-mediated adherence to the intestinal epithelia is a key step in enteroaggregative Escherichia coli (EAEC) pathogenesis. To date, four fimbriae have been described for EAEC; aggregative adherence fimbria II (AAF/II) is the most important adherence factor for EAEC prototype strain 042. Previously, we described results showing that extracellular matrix (ECM) components might be involved in the recognition of AAF/II fimbriae by intestinal cells. In this study, we sought to identify novel potential receptors on intestinal epithelial cells recognized by the AAF/II fimbriae. Purified AafA-dsc protein, the major subunit of AAF/II fimbriae, was incubated with a monolayer of T84 cells, cross-linked to the surface-exposed T84 cell proteins, and immunoprecipitated by using anti-AafA antibodies. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of cellular proteins bound to AafA-dsc protein identified laminin (previously recognized as a potential receptor for AAF/II) and cytokeratin 8 (CK8). Involvement of the major subunit of AAF/II fimbriae (AafA protein) in the binding to recombinant CK8 was confirmed by adherence assays with purified AAF/II fimbriae, AafA-dsc protein, and strain 042. Moreover, HEp-2 cells transfected with CK8 small interfering RNA (siRNA) showed reduced 042 adherence compared with cells transfected with scrambled siRNA as a control. Adherence of 042 to HEp-2 cells preincubated with antibodies against ECM proteins or CK8 was substantially reduced. Altogether, our results supported the idea of a role of CK8 as a potential receptor for EAEC.

Autotransporters but not pAA are critical for rabbit colonization by Shiga toxin-producing Escherichia coli O104:H4

The outbreak of diarrhea and hemolytic uremic syndrome that occurred in Germany in 2011 was caused by a Shiga toxin-producing enteroaggregative Escherichia coli (EAEC) strain. The strain was classified as EAEC due to the presence of a plasmid (pAA) that mediates a characteristic pattern of aggregative adherence on cultured cells, the defining feature of EAEC that has classically been associated with virulence. Here, we describe an infant rabbit-based model of intestinal colonization and diarrhea caused by the outbreak strain, which we use to decipher the factors that mediate the pathogen’s virulence. Shiga toxin is the key factor required for diarrhea. Unexpectedly, we observe that pAA is dispensable for intestinal colonization and development of intestinal pathology. Instead, chromosome-encoded autotransporters are critical for robust colonization and diarrheal disease in this model. Our findings suggest that conventional wisdom linking aggregative adherence to EAEC intestinal colonization is false for at least a subset of strains.

Extended spectrum betalactamase producing Enteroaggregative Escherichia coli from young children in Iran.

The aim of this study was to investigate the frequency of betalactamase producing EAEC isolates among young children with diarrhea in Zanjan, Iran. Entero aggregative Escherichia coli (EAEC) is an emerging enteric pathogen associated with acute and persistent diarrhea and the evolution and spread of acquired extended spectrum betalactamases (ESBLs) among these strains has become a serious problem in the management of infectious diseases in developing countries. During the period from March 2011 to January 2012, 140 isolates of E. coli from diarrheal children aged 0-60 months and 90 isolates from age-matched controls without diarrhea were investigated for EAEC using PCR. Antimicrobial susceptibility testing was performed as CLSI guidelines and betalactamase genes, including bla TEM, bla CTX-M, bla IMP, bla VIM and bla NDM-1 investigated in EAEC isolates. In this study, EAEC was detected with slightly higher frequency in children with (8%) than in children without (4.6%) diarrhea (P > 0.05). Diarrheagenic E. coli exhibited high level resistance to aztreonam (80.7%), amoxicillin (74.4%) and tetracycline (69.3%). Also, 86.4% of E. coli isolates were resistant to at least three different classes of antimicrobial agents and considered as multidrug resistance. Molecular characterization of betalactamase genes showed that bla TEM was the most frequently isolated betalactamase. It was detected in 78.9% of ESBL producing EAEC isolates. Also, the frequency of bla CTX-M was 63.1% (12/19) and 8 (42.1%) isolates carried the bla TEM and bla CTX-M, simultaneously. None MBL producing EAEC was detected in our study. Our results indicate that ESBLs especially bla TEM and bla CTX-M are widespread among EAEC isolates and appropriate surveillance and control measures are essential to prevent further dissemination of betalactamases in our country.

Atypical enteropathogenic Escherichia coli secretes plasmid encoded toxin.

Plasmid encoded toxin (Pet) is a serine protease originally described in enteroaggregative Escherichia coli (EAEC) prototype strain 042 whose entire characterization was essentially obtained from studies performed with the purified toxin. Here we show that Pet is not exclusive to EAEC. Atypical enteropathogenic Escherichia coli (aEPEC) strains, isolated from diarrhea cases, express Pet and its detection in supernatants of infected HEp-2 cells coincides with the appearance of cell damage, which, in turn, were similar to those described with purified Pet. Pet secretion and the cytotoxic effects are time and culture medium dependent. In presence of DMEM supplemented with tryptone cell rounding and detachment were observed after just 5 h of incubation with the bacteria. In the absence of tryptone, the cytotoxic effects were detected only after 24 h of infection. We also show that, in addition to the prototype EAEC, other pet+ EAEC strains, also isolated from diarrhea cases, induce cellular damage in the same degree as the aEPEC. The cytotoxic effects of EAEC and aEPEC strains were significantly reduced in the presence of a serine protease inhibitor or anti-Pet IgG serum. Our results show a common aspect between the aEPEC and EAEC and provide the first evidence pointing to a role of Pet in aEPEC pathogenesis.

Whole genome sequence comparison of vtx2-converting phages from Enteroaggregative Haemorrhagic Escherichia coli strains.

BackgroundEnteroaggregative Haemorrhagic E. coli (EAHEC) is a new pathogenic group of E. coli characterized by the presence of a vtx2-phage integrated in the genomic backbone of Enteroaggregative E. coli (EAggEC). So far, four distinct EAHEC serotypes have been described that caused, beside the large outbreak of infection occurred in Germany in 2011, a small outbreak and six sporadic cases of HUS in the time span 1992–2012. In the present work we determined the whole genome sequence of the vtx2-phage, termed Phi-191, present in the first described EAHEC O111:H2 isolated in France in 1992 and compared it with those of the vtx-phages whose sequences were available.ResultsThe whole genome sequence of the Phi-191 phage was identical to that of the vtx2-phage P13374 present in the EAHEC O104:H4 strain isolated during the German outbreak 20 years later. Moreover, it was also almost identical to those of the other vtx2-phages of EAHEC O104:H4 strains described so far. Conversely, the Phi-191 phage appeared to be different from the vtx2-phage carried by the EAHEC O111:H21 isolated in the Northern Ireland in 2012.The comparison of the vtx2-phages sequences from EAHEC strains with those from the vtx-phages of typical Verocytotoxin-producing E. coli strains showed the presence of a 900 bp sequence uniquely associated with EAHEC phages and encoding a tail fiber.ConclusionsAt least two different vtx2-phages, both characterized by the presence of a peculiar tail fiber-coding gene, intervened in the emergence of EAHEC. The finding of an identical vtx2-phage in two EAggEC strains isolated after 20 years in spite of the high variability described for vtx-phages is unexpected and suggests that such vtx2-phages are kept under a strong selective pressure.The observation that different EAHEC infections have been traced back to countries where EAggEC infections are endemic and the treatment of human sewage is often ineffective suggests that such countries may represent the cradle for the emergence of the EAHEC pathotype. In these regions, EAggEC of human origin can extensively contaminate the environment where they can meet free vtx-phages likely spread by ruminants excreta.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-574) contains supplementary material, which is available to authorized users.

Participation of integrin α5β1 in the fibronectin-mediated adherence of enteroaggregative Escherichia coli to intestinal cells.

Adherence to the intestinal epithelia is a key feature in enteroaggregative Escherichia coli (EAEC) infection. The aggregative adherence fimbriae (AAFs) are involved in EAEC interaction with receptors at the surface of intestinal cells. We and others have demonstrated that fibronectin is a receptor for AAF/II fimbriae. Considering that the major cellular receptor of fibronectin is integrin α5β1, in this study we evaluated the participation of this receptor in the fibronectin-mediated adherence of EAEC strain 042 to intestinal cells. We found that EAEC strain 042 has the ability to bind directly and indirectly to integrin α5β1; direct binding was not mediated by AAF/II fimbriae and indirect binding was mediated by AAF/II and fibronectin. Coimmunoprecipitation assays confirmed the formation of the complex AafA/fibronectin/integrin α5β1. To evaluate EAEC adherence to intestinal cells, T84 cells were incubated with fibronectin and an antibody that blocks the interaction region of integrin α5β1 to fibronectin, the RGD site. Under these conditions, we found the number of adherent bacteria to epithelial cells significantly reduced. Additionally, fibronectin-mediated adherence of EAEC strain 042 was abolished in HEp-2 cells transfected with integrin α5 shRNA. Altogether, our data support the involvement of integrin α5β1 in the fibronectin-mediated EAEC binding to intestinal cells.

Antimicrobial resistance patterns of diarrheagenic and non-diarrheagenic Escherichia coli isolates from Libyan children

Background: Diarrhea continues to be one of the most common causes of morbidity and mortality among infants and children in developing countries. This study was carried out among Libyan children in Benghazi to investigate the prevalence of diarrheagenic E.coli types and antimicrobial susceptibility profiles of E.coli isolates from their stools. Methods & patients: A total of 200 children with diarrhea and 90 without diarrhea were investigated for presence of E.coli in their stools using culture on MacConkey agar, and the isolates were examined for detection diarrheagenic E. coli types, antimicrobial susceptibility pattern, presence of ESBL-CTX-M genes and integrons using PCR . Results: The study indicated that diarrhoeagenic E. coli isolates were found only in stools of children with diarrhoea. The detected diarrhoeagenic E. coli types; were 4 (2%) enteroaggregative E. coli (EAEC), 3 (1.5%) enteroinvasive E. coli (EIEC), and 1(0.5%) enterohemorrhagic E. coli (EHEC), and both enterotoxigenic E. coli (ETEC) and enteropathogenic E. coli (EPEC) were not present. It has also found that multidrug resistant E.coli ( > 3 drugs) was higher prevalent in commensal intestinal E.coli than in diarrheagenic E.coli , and both carried high rates of Class 1 integrons, bla(CTX-M) and fluroquinolone resistance genes . Conclusion: This study revealed the low incidence of diarrhoeagenic E. coli isolates and high prevalent of antimicrobial resistance among normal intestinal E.coli of hospitalized children in Benghazi/Libya.

Emerging Trends in the Etiology and Antimicrobial Susceptibility Pattern of Enteric Pathogens in Rural Coastal India

Introduction: Gastroenteritis is recognized as a serious public health problem in India. It is a syndrome that can be caused by different bacterial, viral and parasitic pathogens. Indiscriminate use of antibacterial agents has resulted in the development of multidrug resistant organisms. A retrospective study was done to analyze the etiological spectrum of diarrhoea and to determine the antimicrobial resistance pattern of bacterial pathogens. Methodology: Fecal specimens from patients with acute or chronic diarrhoea were received prior antibiotic administration and processed for routine microscopy of parasites; culture, antimicrobial susceptibility testing and polymerase chain reaction for bacterial pathogens and latex agglutination for rotavirus. Results: Out of the 6043 stool samples collected during the period of Jan 2005-Dec 2013, 678 (11.2%) enteric pathogens were isolated. The predominant isolates were Diarrheagenic Escherichia coli (DEC) (46.3%) followed by Salmonella, Shigella, Vibrio cholerae and parasites. Certain unusual pathogens were also isolated. Among age wise distribution, Diarrheagenic Escherichia coli (DEC) mainly Enteroaggregative Escherichia coli (EAEC) was isolated from

Extracellular protein biomarkers for the characterization of enterohemorrhagic and enteroaggregative Escherichia coli strains

The extracellular proteins (ECPs) of enterohemorrhagic Escherichia coli (EHEC) can cause hemorrhagic colitis which may cause life threatening hemolytic-uremic syndrome, while that of enteroaggregative E. coli (EAEC) can clump to intestinal membranes. Liquid chromatography–electrospray ionization-tandem mass spectrometry based proteomics is used to evaluate a preliminary study on the extracellular and whole cell protein extracts associated with E. coli strain pathogenicity. Proteomics analysis, which is independent of genomic sequencing, of EAEC O104:H4 (unsequenced genome) identified a number of proteins. Proteomics of EHEC O104:H4, causative agent of the Germany outbreak, showed a closest match with E. coli E55989, in agreement with genomic studies. Dendrogram analysis separated EHEC O157:H7 and EHEC/EAEC O104:H4. ECP analysis compared to that of whole cell processing entails few steps and convenient experimental extraction procedures. Bacterial characterization results are promising in exploring the impact of environmental conditions on E. coli ECP biomarkers with a few relatively straightforward protein extraction steps.

Detection and isolation of Shiga toxin-producing Escherichia coli (STEC) O104 from sprouts

Shiga toxin-producing Escherichia coli (STEC) strains belonging to serogroup O104 have been associated with sporadic cases of illness and have caused outbreaks associated with milk and sprouts. An outbreak that occurred in Europe in 2011 linked to fenugreek sprouts was caused by E. coli O104:H4 that had characteristics of an enteroaggregative E. coli (EAEC) but carried the gene that encoded for Shiga toxin 2. In this study, methods were developed for detection of this enteroaggregative STEC O104, as well as STEC O104 in sprouts. Multiplex PCR assays for enteroaggregative STEC O104:H4 targeted the stx2, aggR, and wzx104 genes, and for STEC O104 targeted the stx1-2, ehxA, and wzx104 genes. After incubating artificially contaminated sprouts at 4°C for 48h and overnight enrichment in modified buffered peptone water with pyruvate supplemented with three antibiotics (mBPWp), the pathogens were detected in all samples inoculated at a level of ca. 100CFU/25g. Several samples inoculated at lower concentrations of ca. 10CFU/25g were negative by the PCR assays, and this could have been due to cells not surviving or not being able to recover after the stress treatment at 4°C for 48h. For isolation of the pathogens, immunomagnetic separation (IMS) using magnetic beads coated with antibodies against O104 were employed, and this was followed by plating the beads onto mRBA and CHROMagar STEC O104 for isolation of E. coli O104:H4 and mRBA and CHROMagar STEC for isolation of E. coli O104:H7. Presumptive colonies were confirmed by agglutination using latex particles attached to antibodies against serogroup O104 and by the multiplex PCR assays. The methodologies described in this study for detection of enteroaggregative STEC O104:H4 and STEC O104 include the use of IMS and latex reagents for serogroup O104, and they enhance the ability to detect and isolate these pathogens from sprouts and potentially other foods, as well.

Slaughterhouse effluent discharges into rivers not responsible for environmental occurrence of enteroaggregative Escherichia coli

Enteroaggregative Shiga-toxin-producing Escherichia coli strains were responsible for a massive outbreak in Europe in 2011, and had been previously isolated from French patients. The objective of this study was to investigate the presence of enteroaggregative E. coli (EAEC) in slaughterhouse effluents (wastewater, slurry, sludge and effluents), and in river waters near these slaughterhouses. A total of 10,618 E. coli isolates were screened by PCR for the presence of EAEC-associated genetic markers (aggR, aap and aatA). None of these markers was detected in E. coli isolated from slaughterhouse samples. A unique enteroaggregative E. coli (EAEC) O126:H8 was detected in river water sampled upstream from slaughterhouse effluent discharge. These results confirmed that animals might not be reservoirs of EAEC, and that further studies are required to evaluate the role of the environment in the transmission of EAEC to humans.