Specific detection of enteroaggregative hemorrhagic Escherichia coli O104:H4 strains by use of the CRISPR locus as a target for a diagnostic real-time PCR.
ABSTRACT: In 2011, a large outbreak of an unusual bacterial strain occurred in Europe. This strain was characterized as a hybrid of an enteroaggregative Escherichia coli (EAEC) and a Shiga toxin-producing E. coli (STEC) strain of the serotype O104:H4. Here, we present a single PCR targeting the clustered regularly interspaced short palindromic repeats locus of E. coli O104:H4 (CRISPR(O104:H4)) for specific detection of EAEC STEC O104:H4 strains from different geographical locations and time periods. The specificity of the CRISPR(O104:H4) PCR was investigated using 1,321 E. coli strains, including reference strains for E. coli O serogroups O1 to O186 and flagellar (H) types H1 to H56. The assay was compared for specificity using PCR assays targeting different O104 antigen-encoding genes (wbwC(O104), wzx(O104), and wzy(O104)). The PCR assays reacted with all types of E. coli O104 strains (O104:H2, O104:H4, O104:H7, and O104:H21) and with E. coli O8 and O9 strains carrying the K9 capsular antigen and were therefore not specific for detection of the EAEC STEC O104:H4 type. A single PCR developed for the CRISPR(O104:H4) target was sufficient for specific identification and detection of the 48 tested EAEC STEC O104:H4 strains. The 35 E. coli O104 strains expressing H types other than H4 as well as 8 E. coli strains carrying a K9 capsular antigen tested all negative for the CRISPR(O104:H4) locus. Only 12 (0.94%) of the 1,273 non-O104:H4 E. coli strains (serotypes Ont:H2, O43:H2, O141:H2, and O174:H2) reacted positive in the CRISPR(O104:H4) PCR (99.06% specificity).
Project description:Ruminants, in particular bovines, are the primary reservoir of Shiga toxin-producing E. coli (STEC), but whole genome analyses of the current German ESBL-producing O104:H4 outbreak strain of sequence type (ST) 678 showed this strain to be highly similar to enteroaggregative E. coli (EAEC). Strains of the EAEC pathotype are basically adapted to the human host. To clarify whether in contrast to this paradigm, the O104:H4 outbreak strain and/or EAEC may also be able to colonize ruminants, we screened a total of 2.000 colonies from faecal samples of 100 cattle from 34 different farms - all located in the HUS outbreak region of Northern Germany - for genes associated with the O104:H4 HUS outbreak strain (stx2, terD, rfbO104, fliCH4), STEC (stx1, stx2, escV), EAEC (pAA, aggR, astA), and ESBL-production (blaCTX-M, blaTEM, blaSHV).The faecal samples contained neither the HUS outbreak strain nor any EAEC. As the current outbreak strain belongs to ST678 and displays an en-teroaggregative and ESBL-producing phenotype, we additionally screened selected strains for ST678 as well as the aggregative adhesion pattern in HEp-2 cells. However, we were unable to find any strains belonging to ST678 or showing an aggregative adhesion pattern. A high percentage of animals (28%) shed STEC, corroborating previous knowl-edge and thereby proving the validity of our study. One of the STEC also harboured the LEE pathogenicity island. In addition, eleven animals shed ESBL-producing E. coli.While we are aware of the limitations of our survey, our data support the theory, that, in contrast to other Shiga-toxin producing E. coli, cattle are not the reservoir for the O104:H4 outbreak strain or other EAEC, but that the outbreak strain seems to be adapted to humans or might have yet another reservoir, raising new questions about the epidemiology of STEC O104:H4.
Project description:Enteroaggregative (EAEC) and Shiga-toxin producing Escherichia coli (STEC) are a major cause of diarrhea worldwide. E. coli carrying both virulence factors characteristic for EAEC and STEC and producing extended-spectrum beta-lactamase caused severe and protracted disease during an outbreak of E. coli O104:H4 in Europe in 2011. We assessed the opportunities for E. coli carrying the aggR and stx genes to emerge in 'backyard' farms in south-east Asia.Faecal samples collected from 204 chicken farms; 204 farmers and 306 age- and gender-matched individuals not exposed to poultry farming were plated on MacConkey agar plates with and without antimicrobials being supplemented. Sweep samples obtained from MacConkey agar plates without supplemented antimicrobials were screened by multiplex PCR for the detection of the stx1, stx2 and aggR genes. One chicken farm sample each (0.5 %) contained the stx1 and the aggR gene. Eleven (2.4 %) human faecal samples contained the stx1 gene, 2 samples (0.4 %) contained stx2 gene, and 31 (6.8 %) contained the aggR gene. From 46 PCR-positive samples, 205 E. coli isolates were tested for the presence of stx1, stx2, aggR, wzx O104 and fliC H4 genes. None of the isolates simultaneously contained the four genetic markers associated with E. coli O104:H4 epidemic strain (aggR, stx2, wzx O104 and fliC H4 ). Of 34 EAEC, 64.7 % were resistant to 3(rd)-generation cephalosporins.These results indicate that in southern Vietnam, the human population is a more likely reservoir of aggR and stx gene carrying E. coli than the chicken population. However, conditions for transmission of isolates and/or genes between human and animal reservoirs resulting in the emergence of highly virulent E. coli strains are still favorable, given the nature of'backyard' farms in Vietnam.
Project description:Shiga toxin 2 (Stx2)-producing Escherichia coli (STEC) O104:H4 caused one of the world's largest outbreaks of hemorrhagic colitis and hemolytic uremic syndrome in Germany in 2011. These strains have evolved from enteroaggregative E. coli (EAEC) by the acquisition of the Stx2 genes and have been designated enteroaggregative hemorrhagic E. coli. Nucleotide sequencing has shown that the Stx2 gene is carried by prophages integrated into the chromosome of STEC O104:H4. We studied the properties of Stx2-encoding bacteriophages which are responsible for the emergence of this new type of E. coli pathogen. For this, we analyzed Stx bacteriophages from STEC O104:H4 strains from Germany (in 2001 and 2011), Norway (2006), and the Republic of Georgia (2009). Viable Stx2-encoding bacteriophages could be isolated from all STEC strains except for the Norwegian strain. The Stx2 phages formed lysogens on E. coli K-12 by integration into the wrbA locus, resulting in Stx2 production. The nucleotide sequence of the Stx2 phage P13374 of a German STEC O104:H4 outbreak was determined. From the bioinformatic analyses of the prophage sequence of 60,894 bp, 79 open reading frames were inferred. Interestingly, the Stx2 phages from the German 2001 and 2011 outbreak strains were found to be identical and closely related to the Stx2 phages from the Georgian 2009 isolates. Major proteins of the virion particles were analyzed by mass spectrometry. Stx2 production in STEC O104:H4 strains was inducible by mitomycin C and was compared to Stx2 production of E. coli K-12 lysogens.
Project description:Intestinal infections represent an important public health concern worldwide. Escherichia coli is one of the main bacterial agents involved in the pathogenesis of different diseases. In 2011, an outbreak of hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) in Germany was related to a non-O157 STEC strain of O104:H4 serotype. The difficulty in identifying the origin of the bacteria related to the outbreak showed the importance of having epidemiological information from different parts of the world. The aim of this study was to perform a retrospective analysis to determine if E. coli strains isolated from cattle from different locations in Mexico have similar characteristics to those isolated in other countries. Samples obtained in different years from 252 cows belonging to 5 herds were analyzed. A total of 1,260 colonies were selected from the 252 samples, 841 (67%) of which corresponded to E. coli and 419 (33%) to other enterobacteria. In total, 78% (656) of the E. coli strains could be serotyped, of which 393 (59.9%) belonged to 5 diarrheagenic (DEC) pathotypes. Serotyping showed STEC (40.7%) and ETEC (26.7%) strains were more common. PCR assays were used to determine the presence of STEC (eae, stx1, stx2, and ehxA) and EAEC (aatA, aggR, and aapA) genes, and phylogenetic groups. The results showed that 70 strains belonging to 23 serogroups were stx1 and stx2 positive, while 13 strains from the O9 serogroup were ehxA, aggR, and eae positive. Phylogenetic analysis showed 58 (82.9%) strains belonged to A and B1 commensal phylogroups and 12 (17.1%) to B2, D and E virulent phylogroups. An assay to evaluate cross-antigenic reactivity in the serum of cattle between K9 capsular antigen and O104 LPS by ELISA showed similar responses against both antigens (p > 0.05). The antimicrobial sensitivity assay of the strains showed resistance to AM, CEP, CXM, TE, SXT, cephalosporins and fluoroquinolones. The results show that cattle are carriers and potential transmitters of STEC and ETEC strains containing virulence genes. Epidemiological retrospective studies in different countries are of great help for identifying virulent bacterial strains with the potential to cause outbreaks that may have epidemiological impact in susceptible countries.
Project description:The emergence of novel pathogens poses a major public health threat causing widespread epidemics in susceptible populations. The Escherichia coli O104:H4 strain implicated in a 2011 outbreak in northern Germany caused the highest frequency of hemolytic uremic syndrome (HUS) and death ever recorded in a single E. coli outbreak. Therefore, it has been suggested that this strain is more virulent than other pathogenic E. coli (e.g., E. coli O157:H7). The E. coli O104:H4 outbreak strain possesses multiple virulence factors from both Shiga toxin (Stx)-producing E. coli (STEC) and enteroaggregative E. coli (EAEC), though the mechanism of pathogenesis is not known. Here, we demonstrate that E. coli O104:H4 produces a stable biofilm in vitro and that in vivo virulence gene expression is highest when E. coli O104:H4 overexpresses genes required for aggregation and exopolysaccharide production, a characteristic of bacterial cells residing within an established biofilm. Interrupting exopolysaccharide production and biofilm formation may therefore represent effective strategies for combating future E. coli O104:H4 infections.
Project description:An ongoing outbreak of exceptionally virulent Shiga toxin (Stx)-producing Escherichia coli O104:H4 centered in Germany, has caused over 830 cases of hemolytic uremic syndrome (HUS) and 46 deaths since May 2011. Serotype O104:H4, which has not been detected in animals, has rarely been associated with HUS in the past. To prospectively elucidate the unique characteristics of this strain in the early stages of this outbreak, we applied whole genome sequencing on the Life Technologies Ion Torrent PGM™ sequencer and Optical Mapping to characterize one outbreak isolate (LB226692) and a historic O104:H4 HUS isolate from 2001 (01-09591). Reference guided draft assemblies of both strains were completed with the newly introduced PGM™ within 62 hours. The HUS-associated strains both carried genes typically found in two types of pathogenic E. coli, enteroaggregative E. coli (EAEC) and enterohemorrhagic E. coli (EHEC). Phylogenetic analyses of 1,144 core E. coli genes indicate that the HUS-causing O104:H4 strains and the previously published sequence of the EAEC strain 55989 show a close relationship but are only distantly related to common EHEC serotypes. Though closely related, the outbreak strain differs from the 2001 strain in plasmid content and fimbrial genes. We propose a model in which EAEC 55989 and EHEC O104:H4 strains evolved from a common EHEC O104:H4 progenitor, and suggest that by stepwise gain and loss of chromosomal and plasmid-encoded virulence factors, a highly pathogenic hybrid of EAEC and EHEC emerged as the current outbreak clone. In conclusion, rapid next-generation technologies facilitated prospective whole genome characterization in the early stages of an outbreak.
Project description:In 2011, a Shiga toxin-producing Enteroaggregative Escherichia coli (EAEC Stx2a+) O104:H4 strain caused a serious outbreak of acute gastroenteritis and hemolytic-uremic syndrome (HUS) in Germany. In 2013, E. coli O104:H4 isolates were obtained from a patient with HUS and her friend showing only gastrointestinal complaints. The antimicrobial resistance and virulence profiles of these isolates together with three EAEC Stx2a+ O104:H4 isolates from 2011 were determined and compared. Whole-genome sequencing (WGS) was performed for detailed characterization and to determine genetic relationship of the isolates. Four additional genomes of EAEC Stx2a+ O104:H4 isolates of 2009 and 2011 available on NCBI were included in the virulence and phylogenetic analysis. All E. coli O104:H4 isolates tested were positive for stx2a, aatA, and terD but were negative for escV. All, except one 2011 isolate, were positive for aggR and were therefore considered EAEC. The EAEC Stx2a+ O104:H4 isolates of 2013 belonged to sequence type (ST) ST678 as the 2011 isolates and showed slightly different resistance and virulence patterns compared to the 2011 isolates. Core-genome phylogenetic analysis showed that the isolates of 2013 formed a separate cluster from the isolates of 2011 and 2009 by 27 and 20 different alleles, respectively. In addition, only a one-allele difference was found between the isolate of the HUS-patient and that of her friend. Our study shows that EAEC Stx2a+ O104:H4 strains highly similar to the 2011 outbreak clone in their core genome are still circulating necessitating proper surveillance to prevent further outbreaks with these potentially pathogenic strains. In addition, WGS not only provided a detailed characterization of the isolates but its high discriminatory power also enabled us to discriminate the 2013 isolates from the isolates of 2009 and 2011 expediting the use of WGS in public health services to rapidly apply proper infection control strategies.
Project description:Various H-serotypes of the Shiga toxin-producing Escherichia coli (STEC) O104, including H4, H7, H21, and H¯, have been associated with sporadic cases of illness and have caused food-borne outbreaks globally. In the U.S., STEC O104:H21 caused an outbreak associated with milk in 1994. However, there is little known on the evolutionary origins of STEC O104 strains, and how genotypic diversity contributes to pathogenic potential of various O104 H-antigen serotypes isolated from different ecological niches and/or geographical regions.Two STEC O104:H21 (milk outbreak strain) and O104:H7 (cattle isolate) strains were shot-gun sequenced, and the genomes were closed. The intimin (eae) gene, involved in the attaching-effacing phenotype of diarrheagenic E. coli, was not found in either strain. Examining various O104 genome sequences, we found that two "complete" left and right end portions of the locus of enterocyte effacement (LEE) pathogenicity island were present in 13 O104 strains; however, the central portion of LEE was missing, where the eae gene is located. In O104:H4 strains, the missing central portion of the LEE locus was replaced by a pathogenicity island carrying the aidA (adhesin involved in diffuse adherence) gene and antibiotic resistance genes commonly carried on plasmids. Enteroaggregative E. coli-specific virulence genes and European outbreak O104:H4-specific stx2-encoding Escherichia P13374 or Escherichia TL-2011c bacteriophages were missing in some of the O104:H4 genome sequences available from public databases. Most of the genomic variations in the strains examined were due to the presence of different mobile genetic elements, including prophages and genomic island regions. The presence of plasmids carrying virulence-associated genes may play a role in the pathogenic potential of O104 strains.The two strains sequenced in this study (O104:H21 and O104:H7) are genetically more similar to each other than to the O104:H4 strains that caused an outbreak in Germany in 2011 and strains found in Central Africa. A hypothesis on strain evolution and pathogenic potential of various H-serotypes of E. coli O104 strains is proposed.
Project description:Escherichia coli O104:H4, a hybrid pathotype reported in a large 2011 foodborne outbreak in Germany, has not been detected in cattle feces. However, cattle harbor and shed in the feces other O104 serotypes, particularly O104:H7, which has been associated with sporadic cases of diarrhea in humans. The objective of our study was to assess the virulence potential of Shiga toxin-producing E. coli (STEC) O104:H7 isolated from feces of feedlot cattle using DNA microarray. Six strains of STEC O104:H7 isolated from cattle feces were analyzed using FDA-E. coli Identification (ECID) DNA microarray to determine their virulence profiles and compare them to the human strains (clinical) of O104:H7, STEC O104:H4 (German outbreak strain), and O104:H21 (milk-associated Montana outbreak strain). Scatter plots were generated from the array data to visualize the gene-level differences between bovine and human O104 strains, and Pearson correlation coefficients (r) were determined. Splits tree was generated to analyze relatedness between the strains. All O104:H7 strains, both bovine and human, similar to O104:H4 and O104:H21 outbreak strains were negative for intimin (eae). The bovine strains were positive for Shiga toxin 1 subtype c (stx1c), enterohemolysin (ehxA), tellurite resistance gene (terD), IrgA homolog protein (iha), type 1 fimbriae (fimH), and negative for genes that code for effector proteins of type III secretory system. The six cattle O104 strains were closely related (r = 0.86-0.98) to each other, except for a few differences in phage related and non-annotated genes. One of the human clinical O104:H7 strains (2011C-3665) was more closely related to the bovine O104:H7 strains (r = 0.81-0.85) than the other four human clinical O104:H7 strains (r = 0.75-0.79). Montana outbreak strain (O104:H21) was more closely related to four of the human clinical O104:H7 strains than the bovine O104:H7 strains. None of the bovine E. coli O104 strains carried genes characteristic of E. coli O104:H4 German outbreak strain and unlike other human strains were also negative for Shiga toxin 2. Because cattle E. coli O104:H7 strains possess stx1c and genes that code for enterohemolysin and a variety of adhesins, the serotype has the potential to be a diarrheagenic foodborne pathogen in humans.
Project description:Escherichia coli O104:H4 was associated with a severe foodborne disease outbreak originating in Germany in May 2011. More than 4000 illnesses and 50 deaths were reported. The outbreak strain was a typical enteroaggregative E. coli (EAEC) that acquired an antibiotic resistance plasmid and a Shiga-toxin 2 (Stx2)-encoding bacteriophage. Based on whole-genome phylogenies, the O104:H4 strain was most closely related to other EAEC strains; however, Stx2-bacteriophage are mobile, and do not necessarily share an evolutionary history with their bacterial host. In this study, we analyzed Stx2-bacteriophage from the E. coli O104:H4 outbreak isolates and compared them to all available Stx2-bacteriophage sequences. We also compared Stx2 production by an E. coli O104:H4 outbreak-associated isolate (ON-2011) to that of E. coli O157:H7 strains EDL933 and Sakai. Among the E. coli Stx2-phage sequences studied, that from O111:H- strain JB1-95 was most closely related phylogenetically to the Stx2-phage from the O104:H4 outbreak isolates. The phylogeny of most other Stx2-phage was largely concordant with their bacterial host genomes. Finally, O104:H4 strain ON-2011 produced less Stx2 than E. coli O157:H7 strains EDL933 and Sakai in culture; however, when mitomycin C was added, ON-2011 produced significantly more toxin than the E. coli O157:H7 strains. The Stx2-phage from the E. coli O104:H4 outbreak strain and the Stx2-phage from O111:H- strain JB1-95 likely share a common ancestor. Incongruence between the phylogenies of the Stx2-phage and their host genomes suggest the recent Stx2-phage acquisition by E. coli O104:H4. The increase in Stx2-production by ON-2011 following mitomycin C treatment may or may not be related to the high rates of hemolytic uremic syndrome associated with the German outbreak strain. Further studies are required to determine whether the elevated Stx2-production levels are due to bacteriophage or E. coli O104:H4 host related factors.