Rita Prager

Epidemic Profile of Shiga-Toxin–Producing Escherichia coli O104:H4 Outbreak in Germany

BACKGROUND We describe an outbreak of gastroenteritis and the hemolytic-uremic syndrome caused by Shiga-toxin-producing Escherichia coli in Germany in May, June, and July, 2011. The consumption of sprouts was identified as the most likely vehicle of infection. METHODS We analyzed data from reports in Germany of Shiga-toxin-producing E. coli gastroenteritis and the hemolytic-uremic syndrome and clinical information on patients presenting to Hamburg University Medical Center (HUMC). An outbreak case was defined as a reported case of the hemolytic-uremic syndrome or of gastroenteritis in a patient infected by Shiga-toxin-producing E. coli, serogroup O104 or serogroup unknown, with an onset of disease during the period from May 1 through July 4, 2011, in Germany. RESULTS A total of 3816 cases (including 54 deaths) were reported in Germany, 845 of which (22%) involved the hemolytic-uremic syndrome. The outbreak was centered in northern Germany and peaked around May 21 to 22. Most of the patients in whom the hemolytic-uremic syndrome developed were adults (88%; median age, 42 years), and women were overrepresented (68%). The estimated median incubation period was 8 days, with a median of 5 days from the onset of diarrhea to the development of the hemolytic-uremic syndrome. Among 59 patients prospectively followed at HUMC, the hemolytic-uremic syndrome developed in 12 (20%), with no significant differences according to sex or reported initial symptoms and signs. The outbreak strain was typed as an enteroaggregative Shiga-toxin-producing E. coli O104:H4, producing extended-spectrum beta-lactamase. CONCLUSIONS In this outbreak, caused by an unusual E. coli strain, cases of the hemolytic-uremic syndrome occurred predominantly in adults, with a preponderance of cases occurring in women. The hemolytic-uremic syndrome developed in more than 20% of the identified cases.

Prospective Genomic Characterization of the German Enterohemorrhagic Escherichia coli O104:H4 Outbreak by Rapid Next Generation Sequencing Technology

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.

Shiga Toxin Gene Loss and Transfer In Vitro and In Vivo during Enterohemorrhagic Escherichia coli O26 Infection in Humans

ABSTRACT Escherichia coli serogroup O26 consists of enterohemorrhagic E. coli (EHEC) and atypical enteropathogenic E. coli (aEPEC). The former produces Shiga toxins (Stx), major determinants of EHEC pathogenicity, encoded by bacteriophages; the latter is Stx negative. We have isolated EHEC O26 from patient stools early in illness and aEPEC O26 from stools later in illness, and vice versa. Intrapatient EHEC and aEPEC isolates had quite similar pulsed-field gel electrophoresis (PFGE) patterns, suggesting that they might have arisen by conversion between the EHEC and aEPEC pathotypes during infection. To test this hypothesis, we asked whether EHEC O26 can lose stx genes and whether aEPEC O26 can be lysogenized with Stx-encoding phages from EHEC O26 in vitro. The stx2 loss associated with the loss of Stx2-encoding phages occurred in 10% to 14% of colonies tested. Conversely, Stx2- and, to a lesser extent, Stx1-encoding bacteriophages from EHEC O26 lysogenized aEPEC O26 isolates, converting them to EHEC strains. In the lysogens and EHEC O26 donors, Stx2-converting bacteriophages integrated in yecE or wrbA. The loss and gain of Stx-converting bacteriophages diversifies PFGE patterns; this parallels findings of similar but not identical PFGE patterns in the intrapatient EHEC and aEPEC O26 isolates. EHEC O26 and aEPEC O26 thus exist as a dynamic system whose members undergo ephemeral interconversions via loss and gain of Stx-encoding phages to yield different pathotypes. The suggested occurrence of this process in the human intestine has diagnostic, clinical, epidemiological, and evolutionary implications.

International outbreak of Salmonella Oranienburg due to German chocolate

BackgroundThis report describes a large international chocolate-associated Salmonella outbreak originating from Germany.MethodsWe conducted epidemiologic investigations including a case-control study, and food safety investigations. Salmonella (S.) Oranienburg isolates were subtyped by the use of pulsed-field gel electrophoresis (PFGE).ResultsFrom 1 October 2001 through 24 March 2002, an estimated excess of 439 S. Oranienburg notifications was registered in Germany. Simultaneously, an increase in S. Oranienburg infections was noted in other European countries in the Enter-net surveillance network. In a multistate matched case-control study in Germany, daily consumption of chocolate (matched odds ratio [MOR]: 4.8; 95% confidence interval [CI]: 1.3–26.5), having shopped at a large chain of discount grocery stores (MOR: 4.2; CI: 1.2–23.0), and consumption of chocolate purchased there (MOR: 5.0; CI: 1.1–47.0) were associated with illness. Subsequently, two brands from the same company, one exclusively produced for that chain, tested positive for S. Oranienburg. In two other European countries and in Canada chocolate from company A was ascertained that also contained S. Oranienburg. Isolates from humans and from chocolates had indistinguishable PFGE profiles. No source or point of contamination was identified. Epidemiological identification of chocolate as a vehicle of infections required two months, and was facilitated by proxy measures.ConclusionsDespite the use of improved production technologies, the chocolate industry continues to carry a small risk of manufacturing Salmonella-containing products. Particularly in diffuse outbreak-settings, clear associations with surrogates of exposure should suffice to trigger public health action. Networks such as Enter-net have become invaluable for facilitating rapid and appropriate management of international outbreaks.

Verotoxinogenic Citrobacter freundii associated with severe gastroenteritis and cases of haemolytic uraemic syndrome in a nursery school: green butter as the infection source.

Abstract A summer outbreak of severe gastroenteritis followed by haemolytic uraemic syndrome (HUS) and thrombotic thrombocytopenic purpura in a nursery school and kindergarten is described. Sandwiches prepared with green butter made with contaminated parsley were the likely vehicle of infection. The parsley originated from an organic garden in which manure of pig origin was used instead of artificial fertilizers. Clonally identical verotoxinogenic Citrobacter freundii were found as causative agents of HUS and gastroenteritis and were also detected on the parsley.

Pork Contaminated with Salmonella enterica Serovar 4,[5],12:i:−, an Emerging Health Risk for Humans

ABSTRACT Salmonella enterica subsp. enterica serovar 4,[5],12:i:− is a monophasic variant of S. enterica serovar Typhimurium (antigenic formula 4,[5],12:i:1,2). Worldwide, especially in several European countries and the United States, it has been reported among the 10 most frequently isolated serovars in pigs and humans. In the study reported here, 148 strains of the monophasic serovar isolated from pigs, pork, and humans in 2006 and 2007 in Germany were characterized by various phenotypic and genotypic methods. This characterization was done in order to investigate their clonality, the prevalence of identical subtypes in pigs, pork, and humans, and the genetic relatedness to other S. enterica serovar Typhimurium subtypes in respect to the pathogenic and resistance gene repertoire. Two major clonal lineages of the monophasic serovar were detected which can be differentiated by their phage types and pulsed-field gel electrophoresis (PFGE) profiles. Seventy percent of the strains tested belonged to definite phage type DT193, and those strains were mainly assigned to PFGE cluster B. Nineteen percent of the strains were typed to phage type DT120 and of these 86% belonged to PFGE cluster A. Sixty-five percent of the isolates of both lineages carried core multiresistance to ampicillin, streptomycin, tetracycline, and sulfamethoxazole encoded by the genes blaTEM1-like, strA-strB, tet(B), and sul2. No correlation to the source of isolation was observed in either lineage. Microarray analysis of 61 S. enterica serovar 4,[5],12:i:− and 20 S. enterica serovar Typhimurium isolates tested determining the presence or absence of 102 representative pathogenicity genes in Salmonella revealed no differences except minor variations in single strains within and between the serovars, e.g., by presence of the virulence plasmid in four strains. Overall the study indicates that in Germany S. enterica serovar 4,[5],12:i:− strains isolated from pig, pork, and human are highly related, showing their transmission along the food chain. Since the pathogenicity gene repertoire is highly similar to that of S. enterica serovar Typhimurium, it is essential that interventions are introduced at the farm level in order to limit human infection.

Salmonella Host Cell Invasion Emerged by Acquisition of a Mosaic of Separate Genetic Elements, Including Salmonella Pathogenicity Island 1 (SPI1), SPI5, and sopE2

Salmonella spp. possess a conserved type III secretion system encoded within the pathogenicity island 1 (SPI1; centisome 63), which mediates translocation of effector proteins into the host cell cytosol to trigger responses such as bacterial internalization. Several translocated effector proteins are encoded in other regions of the Salmonella chromosome. It remains unclear how this complex chromosomal arrangement of genes for the type III apparatus and the effector proteins emerged and how the different effector proteins cooperate to mediate virulence. By Southern blotting, PCR, and phylogenetic analyses of highly diverse Salmonella spp., we show here that effector protein genes located in the core of SPI1 are present in all Salmonella lineages. Surprisingly, the same holds true for several effector protein genes located in distant regions of the Salmonella chromosome, namely, sopB (SPI5, centisome 20), sopD (centisome 64), and sopE2 (centisomes 40 to 42). Our data demonstrate that sopB, sopD, and sopE2, along with SPI1, were already present in the last common ancestor of all contemporary Salmonella spp. Analysis of Salmonella mutants revealed that host cell invasion is mediated by SopB, SopE2, and, in the case of Salmonella enterica serovar Typhimurium SL1344, by SopE: a sopB sopE sopE2-deficient triple mutant was incapable of inducing membrane ruffling and was >100-fold attenuated in host cell invasion. We conclude that host cell invasion emerged early during evolution by acquisition of a mosaic of genetic elements (SPI1 itself, SPI5 [sopB], and sopE2) and that the last common ancestor of all contemporary Salmonella spp. was probably already invasive.

Enterohemorrhagic Escherichia coli in Human Infection: In Vivo Evolution of a Bacterial Pathogen

BACKGROUND Enterohemorrhagic Escherichia coli (EHEC) cause most cases of the hemolytic uremic syndrome (HUS) worldwide. To investigate genetic changes in EHEC during the course of human infection, we analyzed consecutive stool samples and shed isolates from patients with HUS, focusing on the genes encoding Shiga toxin (stx) and intimin (eae). METHODS Sequential stool samples from 210 patients with HUS were investigated for the persistence of E. coli strains harboring stx and/or eae. Initial stool samples were collected during the acute phase of HUS, and subsequent stool samples were collected 3-16 days later (median interval, 8 days). RESULTS Organisms that were stx and eae positive (stx+/eae+ strains; n=137) or stx negative and eae positive (stx-/eae+ strains; n=5) were detected in the initial stool samples from 142 patients. Subsequently, the proportion of those who shed stx+/eae+ strains decreased to 13 of 210 patients, whereas the proportion of those who shed strains that were stx-/eae+ increased to 12 of 210 patients. Seven patients who initially excreted strains that were stx+/eae+ shed, at second analysis, stx-/eae+ strains of the same serotypes; they had no free fecal Shiga toxin at follow-up. Comparison of the initial and follow-up isolates from these patients with use of molecular-epidemiological methods revealed loss of stx genes and genomic rearrangement. CONCLUSIONS We demonstrate the loss of a critical bacterial virulence factor from pathogens during very brief intervals in the human host. These genetic changes have evolutionary, diagnostic, and clinical implications. Generation of stx- mutants might contribute to subclonal evolution and evolutionary success.

Characterization of cytolethal distending toxin genes and expression in shiga toxin-producing Escherichia coli strains of non-O157 serogroups.

ABSTRACT We identified cytolethal distending toxin and its gene (cdt) in 17 of 340 non-O157 Shiga toxin-producing Escherichia coli (STEC) strains (serotypes O73:H18, O91:H21, O113:H21, and O153:H18), all of which were eae negative. cdt is either chromosomal and homologous to cdt-V (serotypes O73:H18, O91:H21, and O113:H21) or plasmidborne and identical to cdt-III (serotype O153:H18). Among eae-negative STEC, cdt was associated with disease (P = 0.003).

Large listeriosis outbreak linked to cheese made from pasteurized milk, Germany, 2006-2007.

A commercial cheese (acid curd) made from pasteurized milk caused a large listeriosis outbreak in Germany from October 2006 through February 2007. The Listeria monocytogenes outbreak strain was identified in humans and in cheese samples from a patients home and from the production plant. During the outbreak period, 189 patients were affected, which was 97% above the mean case number for the respective time period of the years 2002 to 2005. Of patients with available detailed information on cheese consumption (n=47), 70% reported to have consumed the incriminated cheese product. Recent European food safety alerts due to Listeria-contaminated cheeses more often concerned products made from pasteurized or heat-treated milk than from raw milk. The findings should be considered in prevention guidelines addressing vulnerable populations.