Sorbitol-Fermenting Enterohemorrhagic Escherichia coli O157:H− Isolates from Czech Patients with Novel Plasmid Composition Not Previously Seen in German Isolates

ABSTRACT Sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H− strains, first identified in Germany, have emerged as important pathogens throughout Europe. Besides chromosomally encoded Shiga toxin 2a (the major virulence factor), several putative virulence loci, including the hly, etp, and sfp operons, encoding EHEC hemolysin, type II secretion system proteins, and Sfp fimbriae, respectively, are located on the 121-kb plasmid pSFO157 in German strains. Here we report novel SF EHEC O157:H− strains isolated from patients in the Czech Republic. These strains share the core genomes and chromosomal virulence loci encoding toxins (stx 2a and the cdtV-ABC operon) and adhesins (eae-γ, efa1, lpfA O157OI-141, and lpfA O157OI-154) with German strains but differ essentially in their plasmids. In contrast to all previously detected SF EHEC O157:H− strains, the Czech strains carry two plasmids, of 79 kb and 86 kb. The 79-kb plasmid harbors the sfp operon, but neither of the plasmids contains the hly and etp operons. Sequence analyses demonstrated that the 79-kb plasmid (pSFO157 258/98-1) evolved from pSFO157 of German strains by deletion of a 41,534-bp region via homologous recombination, resulting in loss of the hly and etp operons. The 86-kb plasmid (pSFO157 258/98-2) displays 98% sequence similarity to a 92.7-kb plasmid of an extraintestinal pathogenic E. coli bloodstream isolate. Our finding of this novel plasmid composition in SF EHEC O157:H− strains extends the evolutionary history of EHEC O157 plasmids. Moreover, the unique molecular plasmid characteristics permit the identification of such strains, thereby facilitating further investigations of their geographic distribution, clinical significance, and epidemiology. IMPORTANCE Since their first identification in Germany in 1989, sorbitol-fermenting enterohemorrhagic Escherichia coli O157:H− (nonmotile) strains have emerged as important causes of the life-threatening disease hemolytic-uremic syndrome in Europe. They account for 10 to 20% of sporadic cases of this disease and have caused several large outbreaks. The strains isolated throughout Europe share conserved chromosomal and plasmid characteristics. Here we identified novel sorbitol-fermenting enterohemorrhagic E. coli O157:H− patient isolates in the Czech Republic which differ from all such strains reported previously by their unique plasmid characteristics, including plasmid number, composition of plasmid-carried virulence genes, and plasmid origins. Our findings contribute substantially to understanding the evolution of E. coli O157 strains and their plasmids. In practical terms, they enable the identification of strains with these novel plasmid characteristics in patient stool samples and thus the investigation of their roles as human pathogens in other geographic areas.

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Antimicrobial Resistance in Fecal Escherichia coli Isolates from Healthy Urban Children of Two Age Groups in Relation to Their Antibiotic Therapy

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01724-10 ◽

2011 ◽

Vol 55 (6) ◽

pp. 3005-3007 ◽

Cited By ~ 11

Author(s):

Ivan Literak ◽

Radim Petro ◽

Monika Dolejska ◽

Erika Gruberova ◽

Hana Dobiasova ◽

...

Keyword(s):

Escherichia Coli ◽

Czech Republic ◽

Antimicrobial Resistance ◽

Age Groups ◽

Antibiotic Resistant ◽

The Czech Republic ◽

Content Type ◽

E Coli ◽

Significant Difference ◽

Two Age Groups

ABSTRACTThe study was performed in the Czech Republic during 2007 to 2009. OfEscherichia coliisolates from 275 children aged 6 weeks, 36% (n= 177) were resistant to 1 to 7 antibiotics. Of isolates from 253 children aged 6 to 17 years, 24% (n= 205) were resistant to 1 to 5 antibiotics. There was no significant difference in the prevalences of antibiotic-resistantE. coliisolates between these groups of children, even though the consumptions of antibiotics were quite different.

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Plasmid-Mediated Resistance to Cephalosporins and Fluoroquinolones in Various Escherichia coli Sequence Types Isolated from Rooks Wintering in Europe

Applied and Environmental Microbiology ◽

10.1128/aem.02459-14 ◽

2014 ◽

Vol 81 (2) ◽

pp. 648-657 ◽

Cited By ~ 33

Author(s):

Ivana Jamborova ◽

Monika Dolejska ◽

Jiri Vojtech ◽

Sebastian Guenther ◽

Raluca Uricariu ◽

...

Keyword(s):

Escherichia Coli ◽

Czech Republic ◽

European Countries ◽

Beta Lactamase ◽

The Czech Republic ◽

Content Type ◽

Extended Spectrum Beta Lactamase ◽

E Coli ◽

Lactamase Gene ◽

Sequence Types

ABSTRACTExtended-spectrum-beta-lactamase (ESBL)-producing, AmpC beta-lactamase-producing, and plasmid-mediated quinolone resistance (PMQR) gene-positive strains ofEscherichia coliwere investigated in wintering rooks (Corvus frugilegus) from eight European countries. Fecal samples (n= 1,073) from rooks wintering in the Czech Republic, France, Germany, Italy, Poland, Serbia, Spain, and Switzerland were examined. Resistant isolates obtained from selective cultivation were screened for ESBL, AmpC, and PMQR genes by PCR and sequencing. Pulsed-field gel electrophoresis and multilocus sequence typing were performed to reveal their clonal relatedness. In total, from the 1,073 samples, 152 (14%) cefotaxime-resistantE. coliisolates and 355 (33%)E. coliisolates with reduced susceptibility to ciprofloxacin were found. Eighty-two (54%) of these cefotaxime-resistantE. coliisolates carried the following ESBL genes:blaCTX-M-1(n= 39 isolates),blaCTX-M-15(n= 25),blaCTX-M-24(n= 4),blaTEM-52(n= 4),blaCTX-M-14(n= 2),blaCTX-M-55(n= 2),blaSHV-12(n= 2),blaCTX-M-8(n= 1),blaCTX-M-25(n= 1),blaCTX-M-28(n= 1), and an unspecified gene (n= 1). Forty-seven (31%) cefotaxime-resistantE. coliisolates carried theblaCMY-2AmpC beta-lactamase gene. Sixty-two (17%) of theE. coliisolates with reduced susceptibility to ciprofloxacin were positive for the PMQR genesqnrS1(n= 54),qnrB19(n= 4),qnrS1andqnrB19(n= 2),qnrS2(n= 1), andaac(6′)-Ib-cr(n= 1). Eleven isolates from the Czech Republic (n= 8) and Serbia (n= 3) were identified to be CTX-M-15-producingE. coliclone B2-O25b-ST131 isolates. Ninety-one different sequence types (STs) among 191 ESBL-producing, AmpC-producing, and PMQR gene-positiveE. coliisolates were determined, with ST58 (n= 15), ST10 (n= 14), and ST131 (n= 12) predominating. The widespread occurrence of highly diverse ESBL- and AmpC-producing and PMQR gene-positiveE. coliisolates, including the clinically important multiresistant ST69, ST95, ST117, ST131, and ST405 clones, was demonstrated in rooks wintering in various European countries.

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Potentially Human-Pathogenic Escherichia coli O26 in Norwegian Sheep Flocks

Applied and Environmental Microbiology ◽

10.1128/aem.00189-11 ◽

2011 ◽

Vol 77 (14) ◽

pp. 4949-4958 ◽

Cited By ~ 21

Author(s):

C. Sekse ◽

M. Sunde ◽

B.-A. Lindstedt ◽

P. Hopp ◽

T. Bruheim ◽

...

Keyword(s):

Escherichia Coli ◽

Variable Number Tandem Repeat ◽

Variable Number ◽

Human Pathogens ◽

Content Type ◽

Representative Sampling ◽

E Coli ◽

Pathogenic Escherichia Coli ◽

Close Relationship ◽

Large Survey

ABSTRACTA national survey ofEscherichia coliO26 in Norwegian sheep flocks was conducted, using fecal samples to determine the prevalence. In total, 491 flocks were tested, andE. coliO26 was detected in 17.9% of the flocks. One hundred forty-twoE. coliO26 isolates were examined for flagellar antigens (H typing) and four virulence genes, includingstxandeae, to identify possible Shiga toxin-producingE. coli(STEC) and enteropathogenicE. coli(EPEC). Most isolates (129 out of 142) were identified asE. coliO26:H11. They possessedeaeand may have potential as human pathogens, although only a small fraction were identified as STEC O26:H11, giving a prevalence in sheep flocks of only 0.8%. Correspondingly, the sheep flock prevalence of atypical EPEC (aEPEC) O26:H11 was surprisingly high (15.9%). The genetic relationship between theE. coliO26:H11 isolates was investigated by pulsed-field gel electrophoresis (PFGE) and multilocus variable number tandem repeat analysis (MLVA), identifying 63 distinct PFGE profiles and 22 MLVA profiles. Although the MLVA protocol was less discriminatory than PFGE and a few cases of disagreement were observed, comparison by partition mapping showed an overall good accordance between the two methods. A close relationship between a few isolates of aEPEC O26:H11 and STEC O26:H11 was identified, but all theE. coliO26:H11 isolates should be considered potentially pathogenic to humans. The present study consisted of a representative sampling of sheep flocks from all parts of Norway. This is the first large survey of sheep flocks focusing onE. coliO26 in general, including results of STEC, aEPEC, and nonpathogenic isolates.

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Colonization of Arabidopsis thaliana with Salmonella enterica and Enterohemorrhagic Escherichia coli O157:H7 and Competition by Enterobacter asburiae

Applied and Environmental Microbiology ◽

10.1128/aem.69.8.4915-4926.2003 ◽

2003 ◽

Vol 69 (8) ◽

pp. 4915-4926 ◽

Cited By ~ 205

Author(s):

Michael B. Cooley ◽

William G. Miller ◽

Robert E. Mandrell

Keyword(s):

Escherichia Coli ◽

Arabidopsis Thaliana ◽

Salmonella Enterica ◽

Fluorescent Protein ◽

Enterohemorrhagic Escherichia Coli ◽

Plant Responses ◽

Human Pathogens ◽

E Coli ◽

Green Fluorescent ◽

Enterobacter Asburiae

ABSTRACT Enteric pathogens, such as Salmonella enterica and Escherichia coli O157:H7, have been shown to contaminate fresh produce. Under appropriate conditions, these bacteria will grow on and invade the plant tissue. We have developed Arabidopsis thaliana (thale cress) as a model system with the intention of studying plant responses to human pathogens. Under sterile conditions and at 100% humidity, S. enterica serovar Newport and E. coli O157:H7 grew to 109 CFU g−1 on A. thaliana roots and to 2 × 107 CFU g−1 on shoots. Furthermore, root inoculation led to contamination of the entire plant, indicating that the pathogens are capable of moving on or within the plant in the absence of competition. Inoculation with green fluorescent protein-labeled S. enterica and E. coli O157:H7 showed invasion of the roots at lateral root junctions. Movement was eliminated and invasion decreased when nonmotile mutants of S. enterica were used. Survival of S. enterica serovar Newport and E. coli O157:H7 on soil-grown plants declined as the plants matured, but both pathogens were detectable for at least 21 days. Survival of the pathogen was reduced in unautoclaved soil and amended soil, suggesting competition from indigenous epiphytes from the soil. Enterobacter asburiae was isolated from soil-grown A. thaliana and shown to be effective at suppressing epiphytic growth of both pathogens under gnotobiotic conditions. Seed and chaff harvested from contaminated plants were occasionally contaminated. The rate of recovery of S. enterica and E. coli O157:H7 from seed varied from undetectable to 19% of the seed pools tested, depending on the method of inoculation. Seed contamination by these pathogens was undetectable in the presence of the competitor, Enterobacter asburiae. Sampling of 74 pools of chaff indicated a strong correlation between contamination of the chaff and seed (P = 0.025). This suggested that contamination of the seed occurred directly from contaminated chaff or by invasion of the flower or silique. However, contaminated seeds were not sanitized by extensive washing and chlorine treatment, indicating that some of the bacteria reside in a protected niche on the seed surface or under the seed coat.

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Antibiotic-Mediated Modulations of Outer Membrane Vesicles in Enterohemorrhagic Escherichia coli O104:H4 and O157:H7

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00937-17 ◽

2017 ◽

Vol 61 (9) ◽

Cited By ~ 19

Author(s):

Andreas Bauwens ◽

Lisa Kunsmann ◽

Helge Karch ◽

Alexander Mellmann ◽

Martina Bielaszewska

Keyword(s):

Escherichia Coli ◽

Outer Membrane ◽

Shiga Toxin ◽

Membrane Vesicles ◽

Polymyxin B ◽

Outer Membrane Vesicles ◽

Enterohemorrhagic Escherichia Coli ◽

Content Type ◽

E Coli ◽

Major Virulence Factor

ABSTRACT Ciprofloxacin, meropenem, fosfomycin, and polymyxin B strongly increase production of outer membrane vesicles (OMVs) in Escherichia coli O104:H4 and O157:H7. Ciprofloxacin also upregulates OMV-associated Shiga toxin 2a, the major virulence factor of these pathogens, whereas the other antibiotics increase OMV production without the toxin. These two effects might worsen the clinical outcome of infections caused by Shiga toxin-producing E. coli. Our data support the existing recommendations to avoid antibiotics for treatment of these infections.

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Faecal shedding of verotoxigenic Escherichia coli in cattle in the Czech Republic

Veterinární Medicína ◽

10.17221/3152-vetmed ◽

2011 ◽

Vol 56 (No. 4) ◽

pp. 149-155 ◽

Cited By ~ 4

Author(s):

P. Alexa ◽

L. Konstantinova ◽

Z. Sramkova-Zajacova

Keyword(s):

Escherichia Coli ◽

Czech Republic ◽

Dairy Cattle ◽

Age Groups ◽

Immunomagnetic Separation ◽

The Other ◽

Somatic Antigen ◽

The Czech Republic ◽

E Coli ◽

One Year

A survey to estimate the prevalence of verotoxigenic E. coli (VTEC) or enterohaemorrhagic E. coli (EHEC) in rectal swabs from healthy dairy cattle aged three weeks, three months and one year was conducted in three herds from the Czech Republic. Screening for the presence of the stx1, stx2 and eaeA genes in faecal swab cultures was performed by PCR, and in positive samples, isolated colonies were examined. Immunomagnetic separation was used for the isolation of the VTEC serogroup O157 from samples. VTEC were detected in animals from all three herds under study. In the group of 3-week-old calves, VTEC were only detected in samples collected in the summer months. However, in the other age-groups, VTEC were detected in both the summer and winter months. EHEC shedding was observed in 30 to 100% of the total samples collected from cattle aged three months and one year in the summer months, and in 30 to 60% of samples taken in the winter months. EHEC strains of serogroup O157 were detected in two herds. The range of verotoxins shed by VTEC isolates of serogroup O157 differed between herds. Besides serogroup O157, additional EHEC belonging to the antigen groups O26, O103, O128 and O153 have been identified, and in some of them, no somatic antigen was detected.

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Enterohemorrhagic Escherichia coli as Causes of Hemolytic Uremic Syndrome in the Czech Republic

PLoS ONE ◽

10.1371/journal.pone.0073927 ◽

2013 ◽

Vol 8 (9) ◽

pp. e73927 ◽

Cited By ~ 22

Author(s):

Monika Marejková ◽

Květa Bláhová ◽

Jan Janda ◽

Angelika Fruth ◽

Petr Petráš

Keyword(s):

Escherichia Coli ◽

Czech Republic ◽

Hemolytic Uremic Syndrome ◽

Enterohemorrhagic Escherichia Coli ◽

The Czech Republic ◽

Uremic Syndrome

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Fitness of Enterohemorrhagic Escherichia coli (EHEC)/Enteroaggregative E. coli O104:H4 in Comparison to That of EHEC O157: Survival Studies in Food andIn Vitro

Applied and Environmental Microbiology ◽

10.1128/aem.01796-16 ◽

2016 ◽

Vol 82 (21) ◽

pp. 6326-6334 ◽

Cited By ~ 5

Author(s):

Christina Böhnlein ◽

Jan Kabisch ◽

Diana Meske ◽

Charles M. A. P. Franz ◽

Rohtraud Pichner

Keyword(s):

Escherichia Coli ◽

Meat Product ◽

Enterohemorrhagic Escherichia Coli ◽

Content Type ◽

E Coli ◽

Adverse Conditions ◽

Log Reduction ◽

Fermented Sausages ◽

Survival Studies

ABSTRACTIn 2011, one of the world's largest outbreaks of hemolytic-uremic syndrome (HUS) occurred, caused by a rareEscherichia coliserotype, O104:H4, that shared the virulence profiles of Shiga toxin-producingE. coli(STEC)/enterohemorrhagicE. coli(EHEC) and enteroaggregativeE. coli(EAEC). The persistence and fitness factors of the highly virulent EHEC/EAEC O104:H4 strain, grown either in food orin vitro, were compared with those ofE. coliO157 outbreak-associated strains. The log reduction rates of the different EHEC strains during the maturation of fermented sausages were not significantly different. Both the O157:NM and O104:H4 serotypes could be shown by qualitative enrichment to be present after 60 days of sausage storage. Moreover, the EHEC/EAEC O104:H4 strain appeared to be more viable thanE. coliO157:H7 under conditions of decreased pH and in the presence of sodium nitrite. Analysis of specific EHEC strains in experiments with an EHEC inoculation cocktail showed a dominance of EHEC/EAEC O104:H4, which could be isolated from fermented sausages for 60 days. Inhibitory activities of EHEC/EAEC O104:H4 toward severalE. colistrains, including serotype O157 strains, could be determined. Our study suggests that EHEC/EAEC O104:H4 is well adapted to the multiple adverse conditions occurring in fermented raw sausages. Therefore, it is strongly recommended that STEC strain cocktails composed of several serotypes, instead ofE. coliO157:H7 alone, be used in food risk assessments. The enhanced persistence of EHEC/EAEC O104:H4 as a result of its robustness, as well as the production of bacteriocins, may account for its extraordinary virulence potential.IMPORTANCEIn 2011, a severe outbreak caused by an EHEC/EAEC serovar O104:H4 strain led to many HUS sequelae. In this study, the persistence of the O104:H4 strain was compared with those of other outbreak-relevant STEC strains under conditions of fermented raw sausage production. Both O157:NM and O104:H4 strains could survive longer during the production of fermented sausages thanE. coliO157:H7 strains.E. coliO104:H4 was also shown to be well adapted to the multiple adverse conditions encountered in fermented sausages, and the secretion of a bacteriocin may explain the competitive advantage of this strain in an EHEC strain cocktail. Consequently, this study strongly suggests that enhanced survival and persistence, and the presumptive production of a bacteriocin, may explain the increased virulence of the O104:H4 outbreak strain. Furthermore, this strain appears to be capable of surviving in a meat product, suggesting that meat should not be excluded as a source of potentialE. coliO104:H4 infection.

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Evaluation of Aerated Steam Treatment of Alfalfa and Mung Bean Seeds To Eliminate High Levels of Escherichia coli O157:H7 and O178:H12, Salmonella enterica, and Listeria monocytogenes

Applied and Environmental Microbiology ◽

10.1128/aem.00443-13 ◽

2013 ◽

Vol 79 (15) ◽

pp. 4613-4619 ◽

Cited By ~ 14

Author(s):

Patrick Studer ◽

Werner E. Heller ◽

Jörg Hummerjohann ◽

David Drissner

Keyword(s):

Escherichia Coli ◽

Listeria Monocytogenes ◽

Salmonella Enterica ◽

Mung Bean ◽

Germination Rate ◽

Steam Treatment ◽

Heat Sensitivity ◽

Human Pathogens ◽

Content Type ◽

E Coli

ABSTRACTSprouts contaminated with human pathogens are able to cause food-borne diseases due to the favorable growth conditions for bacteria during germination and because of minimal processing steps prior to consumption. We have investigated the potential of hot humid air, i.e., aerated steam, to treat alfalfa and mung bean seeds which have been artificially contaminated withEscherichia coliO157:H7,Salmonella entericasubsp.entericaserovar Weltevreden, andListeria monocytogenesScott A. In addition, a recently collectedE. coliO178:H12 isolate, characterized by a reduced heat sensitivity, was exposed to the treatment described. Populations ofE. coliO157:H7 andS. entericaon alfalfa and mung bean seeds could be completely eliminated by a 300-s treatment with steam at 70 ± 1°C as revealed by enrichment studies.L. monocytogenesandE. coliO178:H12 could not be completely eliminated from artificially inoculated seeds. However, bacterial populations were reduced by more than 5 log CFU/g on alfalfa and by more than 4 log CFU/g on mung bean seeds. The germination rate of mung beans was not affected by the 300-s treatment compared to the germination rate of untreated seeds whereas that of alfalfa seeds was significantly lower by 11.9%. This chemical-free method is an effective alternative to the 20,000-ppm hypochlorite treatment presently recommended by the U.S. Food and Drug Administration (FDA).

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Occurrence of Potentially Human-Pathogenic Escherichia coli O103 in Norwegian Sheep

Applied and Environmental Microbiology ◽

10.1128/aem.01825-13 ◽

2013 ◽

Vol 79 (23) ◽

pp. 7502-7509 ◽

Cited By ~ 10

Author(s):

Camilla Sekse ◽

Marianne Sunde ◽

Petter Hopp ◽

Torkjel Bruheim ◽

Kofitsyo Sewornu Cudjoe ◽

...

Keyword(s):

Escherichia Coli ◽

Human Pathogens ◽

Outbreak Strain ◽

Banding Patterns ◽

Sheep Population ◽

Content Type ◽

E Coli ◽

Pathogenic Escherichia Coli ◽

Uremic Syndrome ◽

Low Prevalence

ABSTRACTThe investigation of an outbreak of hemorrhagic-uremic syndrome in Norway in 2006 indicated that the outbreak strainEscherichia coliO103:H25 could originate from sheep. A national survey of the Norwegian sheep population was performed, with the aim of identifying and describing a possible reservoir of potentially human-pathogenicE. coliO103, in particular of the H types 2 and 25. The investigation of fecal samples from 585 sheep flocks resulted in 1,222E. coliO103 isolates that were analyzed for the presence ofeaeandstxgenes, while a subset of 369 isolates was further examined for flagellar antigens (H typing),stxsubtypes,bfpA,astA, and molecular typing by pulsed-field gel electrophoresis (PFGE). The total ovineE. coliO103 serogroup was genetically diverse by numbers of H types, virulotypes, and PFGE banding patterns identified, although a tendency of clustering toward serotypes was seen. The flocks positive for potentially human-pathogenicE. coliO103 were geographically widely distributed, and no association could be found with county or geographical region. The survey showed thateae-negative,stx-negativeE. coliO103, probably nonpathogenic to humans, is very common in sheep, with 27.5% of flocks positive. Moreover, the study documented a low prevalence (0.7%) of potentially human-pathogenic Shiga toxin-producingE. coliO103:H2, while STEC O103:H25 was not detected. However, 3.1% and 5.8% of the flocks were positive for enteropathogenicE. coliO103 belonging to H types 2 and 25, respectively. These isolates are of concern as potential human pathogens by themselves but more importantly as possible precursors for human-pathogenic STEC.

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