Angelika Miko

Identification of human-pathogenic strains of Shiga toxin-producing Escherichia coli from food by a combination of serotyping and molecular typing of Shiga toxin genes.

ABSTRACT We examined 219 Shiga toxin-producing Escherichia coli (STEC) strains from meat, milk, and cheese samples collected in Germany between 2005 and 2006. All strains were investigated for their serotypes and for genetic variants of Shiga toxins 1 and 2 (Stx1 and Stx2). stx1 or variant genes were detected in 88 (40.2%) strains and stx2 and variants in 177 (80.8%) strains. Typing of stx genes was performed by stx-specific PCRs and by analysis of restriction fragment length polymorphisms (RFLP) of PCR products. Major genotypes of the Stx1 (stx1, stx1c, and stx1d) and the Stx2 (stx2, stx2d, stx2-O118, stx2e, and stx2g) families were detected, and multiple types of stx genes coexisted frequently in STEC strains. Only 1.8% of the STEC strains from food belonged to the classical enterohemorrhagic E. coli (EHEC) types O26:H11, O103:H2, and O157:H7, and only 5.0% of the STEC strains from food were positive for the eae gene, which is a virulence trait of classical EHEC. In contrast, 95 (43.4%) of the food-borne STEC strains carried stx2 and/or mucus-activatable stx2d genes, an indicator for potential high virulence of STEC for humans. Most of these strains belonged to serotypes associated with severe illness in humans, such as O22:H8, O91:H21, O113:H21, O174:H2, and O174:H21. stx2 and stx2d STEC strains were found frequently in milk and beef products. Other stx types were associated more frequently with pork (stx2e), lamb, and wildlife meat (stx1c). The combination of serotyping and stx genotyping was found useful for identification and for assignment of food-borne STEC to groups with potential lower and higher levels of virulence for humans.

Assessment of Shiga toxin-producing Escherichia coli isolates from wildlife meat as potential pathogens for humans.

ABSTRACT A total of 140 Shiga toxin-producing Escherichia coli (STEC) strains from wildlife meat (deer, wild boar, and hare) isolated in Germany between 1998 and 2006 were characterized with respect to their serotypes and virulence markers associated with human pathogenicity. The strains grouped into 38 serotypes, but eight O groups (21, 146, 128, 113, 22, 88, 6, and 91) and four H types (21, 28, 2, and 8) accounted for 71.4% and 75.7% of all STEC strains from game, respectively. Eighteen of the serotypes, including enterohemorrhagic E. coli (EHEC) O26:[H11] and O103:H2, were previously found to be associated with human illness. Genes linked to high-level virulence for humans (stx2, stx2d, and eae) were present in 46 (32.8%) STEC strains from game. Fifty-four STEC isolates from game belonged to serotypes which are frequently found in human patients (O103:H2, O26:H11, O113:H21, O91:H21, O128:H2, O146:H21, and O146:H28). These 54 STEC isolates were compared with 101 STEC isolates belonging to the same serotypes isolated from farm animals, from their food products, and from human patients. Within a given serotype, most STEC strains were similar with respect to their stx genotypes and other virulence attributes, regardless of origin. The 155 STEC strains were analyzed for genetic similarity by XbaI pulsed-field gel electrophoresis. O103:H2, O26:H11, O113:H21, O128:H2, and O146:H28 STEC isolates from game were 85 to 100% similar to STEC isolates of the same strains from human patients. By multilocus sequence typing, game EHEC O103:H2 strains were attributed to a clonal lineage associated with hemorrhagic diseases in humans. The results from our study indicate that game animals represent a reservoir for and a potential source of human pathogenic STEC and EHEC strains.

Evaluation of Major Types of Shiga Toxin 2e-Producing Escherichia coli Bacteria Present in Food, Pigs, and the Environment as Potential Pathogens for Humans

ABSTRACT Shiga toxin 2e (Stx2e)-producing strains from food (n = 36), slaughtered pigs (n = 25), the environment (n = 21), diseased pigs (n = 19), and humans (n = 9) were investigated for production of Stx2e by enzyme-linked immunosorbent assay, for virulence markers by PCR, and for their serotypes to evaluate their role as potential human pathogens. Stx2e production was low in 64% of all 110 strains. Stx2e production was inducible by mitomycin C but differed considerably between strains. Analysis by nucleotide sequencing and transcription of stx2e genes in high- and low-Stx2e-producing strains showed that toxin production correlated with transcription rates of stx2e genes. DNA sequences specific for the int, Q, dam, and S genes of the stx2e bacteriophage P27 were found in 109 strains, indicating cryptic P27-like prophages, although 102 of these were not complete for all genes tested. Genes encoding intimin (eae), enterohemorrhagic Escherichia coli hemolysin (ehx), or other stx1 or stx2 variants were not found, whereas genes for heat-stable enterotoxins STI, STII, or EAST1 were present in 54.5% of the strains. Seven major serotypes that were associated with diseased pigs (O138:H14, O139:H1, and O141:H4) or with slaughter pigs, food, and the environment (O8:H4, O8:H9, O100:H30, and O101:H9) accounted for 60% of all Stx2e strains. The human Stx2e isolates did not belong to these major serotypes of Stx2e strains, and high production of Stx2e in human strains was not related to diarrheal disease. The results from this study and other studies do not point to Stx2e as a pathogenicity factor for diarrhea and hemolytic uremic syndrome in humans.

Molecular characterization of multiresistant d-tartrate-positive Salmonella enterica serovar paratyphi B isolates.

ABSTRACT Since 1996, the National Salmonella Reference Laboratory of Germany has received an increasing number of Salmonella enterica subsp. enterica serovar Paratyphi B isolates. Nearly all of these belonged to the dextrorotatory tartrate-positive variant (S. enterica subsp. enterica serovar Paratyphi B dT+), formerly called S. enterica subsp. enterica serovar Java. A total of 55 selected contemporary and older S. enterica subsp. enterica serovar Paratyphi B dT+ isolates were analyzed by plasmid profiling, antimicrobial resistance testing, pulsed-field gel electrophoresis, IS200 profiling, and PCR-based detection of integrons. The results showed a high genetic heterogeneity among 10 old strains obtained from 1960 to 1993. In the following years, however, new distinct multiresistant S. enterica subsp. enterica serovar Paratyphi B dT+ clones emerged, and one clonal lineage successfully displaced the older ones. Since 1994, 88% of the isolates investigated were multiple drug resistant. Today, a particular clone predominates in some German poultry production lines, poultry products, and various other sources. It was also detected in contemporary isolates from two neighboring countries as well.

Multiple-Drug Resistance in d-Tartrate-Positive Salmonella enterica Serovar Paratyphi B Isolates from Poultry Is Mediated by Class 2 Integrons Inserted into the Bacterial Chromosome

ABSTRACT The presence of integrons in 85 multiresistant German isolates of the predominating Salmonella enterica subsp. enterica serovar Paratyphi B dT+ clone was investigated. All isolates possessed a chromosomally located Tn7-like class 2 integron carrying the same dfrA1-sat1-aadA1 array of gene cassettes. Only four isolates (4.7%) revealed an additional class 1 integron with two strains each containing the aadA1 or dfrA1-aadA1 gene cassettes.

Spread of a distinct Stx2-encoding phage prototype among Escherichia coli O104:H4 strains from outbreaks in Germany, Norway, and Georgia.

ABSTRACT Shiga toxin 2 (Stx2)-producing Escherichia coli (STEC) O104:H4 caused one of the worlds 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.

Genotypes and virulence characteristics of Shiga toxin-producing Escherichia coli O104 strains from different origins and sources

Sixty-two Escherichia coli strains carrying the wzxO104-gene from different sources, origins and time periods were analyzed for their serotypes, virulence genes and compared for genomic similarity by pulsed-field gel-electrophoresis (PFGE). The O104 antigen was present in 55 strains and the structurally and genetically related capsular antigen K9 in five strains. The presence of 49 genes associated with enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC) and enterohemorrhagic E. coli (EHEC) was investigated. Fifty-four strains of serotypes O104:H2 (n=1), O104:H4 (n=37), O104:H7 (n=5) and O104:H21 (n=11) produced Shiga-toxins (Stx). Among STEC O104, a close association between serotype, virulence gene profile and genomic similarity was found. EAEC virulence genes were only present in STEC O104:H4 strains. EHEC-O157 plasmid-encoded genes were only found in STEC O104:H2, O104:H7 and O104:H21 strains. None of the 62 O104 or K9 strains carried an eae-gene involved in the attaching and effacing phenotype. The 38 O104:H4 strains formed a single PFGE-cluster (>83.7% similarity). Thirty-one of these strains were from the European O104:H4 outbreak in 2011. The outbreak strains and older O104:H4 strains from Germany (2001), Georgia and France (2009) clustered together at>86.2% similarity. O104:H4 strains isolated between 2001 and 2009 differed for some plasmid-encoded virulence genes compared to the outbreak strains from 2011. STEC O104:H21 and STEC O104:H7 strains isolated in the U.S. and in Europe showed characteristic differences in their Stx-types, virulence gene and PFGE profiles indicating that these have evolved separately. E. coli K9 strains were not associated with virulence and were heterogeneous for their serotypes and PFGE profiles.

Emerging types of Shiga toxin-producing E. coli (STEC) O178 present in cattle, deer, and humans from Argentina and Germany

More than 400 serotypes of Shiga toxin-producing Escherichia coli (STEC) have been implicated in outbreaks and sporadic human diseases. In recent years STEC strains belonging to serogroup O178 have been commonly isolated from cattle and food of bovine origin in South America and Europe. In order to explore the significance of these STEC strains as potential human pathogens, 74 German and Argentinean E. coli O178 strains from animals, food and humans were characterized phenotypically and investigated for their serotypes, stx-genotypes and 43 virulence-associated markers by a real-time PCR-microarray. The majority (n = 66) of the O178 strains belonged to serotype O178:H19. The remaining strains divided into O178:H7 (n = 6), O178:H10 (n = 1), and O178:H16 (n = 1). STEC O178:H19 strains were mainly isolated from cattle and food of bovine origin, but one strain was from a patient with hemolytic uremic syndrome (HUS). Genotyping of the STEC O178:H19 strains by pulsed-field gel electrophoresis revealed two major clusters of genetically highly related strains which differ in their stx-genotypes and non-Stx putative virulence traits, including adhesins, toxins, and serine-proteases. Cluster A-strains including the HUS-strain (n = 35) carried genes associated with severe disease in humans (stx2a, stx2d, ehxA, saa, subAB1, lpfAO113, terE combined with stx1a, espP, iha). Cluster B-strains (n = 26) showed a limited repertoire of virulence genes (stx2c, pagC, lpfAO113, espP, iha). Among O178:H7 strains isolated from deer meat and patients with uncomplicated disease a new STEC variant was detected that is associated with the genotype stx1c/stx2b/ehxA/subAB2/espI/[terE]/espP/iha. None of the STEC O178 strains was positive for locus of enterocyte effacement (LEE)- and nle-genes. Results indicate that STEC O178:H19 strains belong to the growing group of LEE-negative STEC that should be considered with respect to their potential to cause diseases in humans.

Ergebnisse, Schlussfolgerungen und Empfehlungen aus zwei Ringversuchen zum Nachweis und zur Isolierung von Shiga (Vero) Toxin bildenden Escherichia coli (STEC) aus Hackfleischproben

ZusammenfassungZur Qualitätssicherung wurden vom Nationalen Referenzlabor für Escherichia coli am BfR in den Jahren 2008 und 2009 zwei Ringversuche (RV2008 und RV2009) zum Nachweis und zur Isolierung von Shiga Toxin bildenden Escherichia coli (STEC) aus Hackfleischproben durchgeführt. Am RV2008 beteiligten sich 23, am RV2009 26 Einrichtungen, die sich mit der mikrobiologischen Kontrolle von Lebensmitteln befassen. Jeder Teilnehmer erhielt zur Analytik fünf Hackfleischproben mit STEC und vier ohne STEC. Im Vergleich konnte im RV2009 eine deutliche Verbesserung der Spezifität und Sensitivität beobachtet werden. Im RV2008 hatten nur vier (17,4%) Teilnehmer alle STEC-positiven Proben identifiziert, im RV2009 waren es 14 (53,8%). Die Zahl der falsch-positiven Befunde sank von 17,4% (RV2008) auf 3,8% (RV2009). Die Ermittlung des Konkordanz odds ratio Wertes zeigte signifikante Unterschiede zwischen den teilnehmenden Laboren. Für die Analyse verwendeten die Teilnehmer überwiegend Methoden in Anlehnung an die Amtlichen Untersuchungsverfahren nach § 64 LFGB, wie Stx-ELISA und Kolonie-Immunoblot, oder stx-PCR und Kolonie-DNA-Hybridisierung. Die auffällig niedrige Sensitivität bei Einsatz von Stx-ELISA im RV2008 konnte auf ein kommerzielles Produkt (Novitek Veterinär), das einige Varianten von Stx1 und Stx2 nicht erkannte, zurückgeführt werden. Es wird empfohlen, nur von unabhängiger Seite evaluierte Nachweisverfahren einzusetzen, um die Spezifität und Sensitivität des Testverfahrens zu gewährleisten. Real-Time stx-PCR als neue Methode wurde verstärkt eingesetzt und erwies sich als viel versprechend. Der Nachweis von enterohämorrhagischen E. coli (EHEC) O157:[H7] als häufigster Erreger von Hämorrhagischer Colitis (HC) und Hämolytisch Urämischem Syndrom (HUS) sollte durch Einführung spezifischer Verfahren (ISO16654) verbessert werden, da nur zwei von 26 Teilnehmern diesen Keim aus einer Hackfleischprobe, die zwei STEC Stämme enthielt, isoliert hatten.AbstractFor quality assurance the National Reference Laboratory for Escherichia coli at the Federal Institute for Risk Assessment had organized two ring trials in 2008 and 2009 (called RV2008 and RV2009) on the detection and isolation of Shiga toxin producing Escherichia coli (STEC) from minced beef samples. There were 23 (RV2008) and 26 (RV2009) participants from institutions dealing with the microbiological control of food. For the analysis, the participants received five samples of minced meat containing STEC and four samples without STEC. By comparing the results, a significant improvement of detection sensitivity and specificity was observed for RV2009. In RV2008, only 4 (17.4%) of the participants had identified all STEC-positive samples, compared to 14 (53.8%) in RV2009. The number of false-positive findings decreased from 17.4% (RV2008) to 3.8% (RV2009). Statistically significant differences between the participating laboratories were found by determination of concordance odds ratios. Most participants used methods for detection and isolation of STEC based on protocols that are officially recommended in Germany (§64 LFGB), such as Shiga toxin (Stx) ELISA/Colony Immunoblot, or stx-PCR/DNA colony hybridization. A strikingly low sensitivity when using the Stx-ELISA as detection method was observed for RV2008. This finding could be attributed to the use of a commercial Stx-ELISA (Novitek Veterinär) that showed deficiencies for identification of Stx1 and some variants of Stx2. It is recommended to use only detection systems that have been independently evaluated for their specificity and sensitivity. Real-time stx-PCR as a new method has been increasingly used and proved to be promising. The detection of enterohaemorrhagic E. coli O157: [H7] as the most common cause of Haemorrhagic Colitis (HC) and Haemolytic Uraemic Syndrome (HUS) should be improved by introduction of specific procedures (ISO16654), since only 2 of 26 participants successfully isolated this agent from a minced meat sample that contained two STEC strains.

Ergebnisse, Schlussfolgerungen und Empfehlungen aus zwei Ringversuchen zum Nachweis und zur Isolierung von Shiga (Vero) Toxin bildenden Escherichia coli (STEC) aus Hackfleischproben@@@Results, conclusions, and recommendations of two ring trials for the detection and isolation of shiga (Vero) toxin producing Escherichia coli (STEC) from minced beef samples

ZusammenfassungZur Qualitätssicherung wurden vom Nationalen Referenzlabor für Escherichia coli am BfR in den Jahren 2008 und 2009 zwei Ringversuche (RV2008 und RV2009) zum Nachweis und zur Isolierung von Shiga Toxin bildenden Escherichia coli (STEC) aus Hackfleischproben durchgeführt. Am RV2008 beteiligten sich 23, am RV2009 26 Einrichtungen, die sich mit der mikrobiologischen Kontrolle von Lebensmitteln befassen. Jeder Teilnehmer erhielt zur Analytik fünf Hackfleischproben mit STEC und vier ohne STEC. Im Vergleich konnte im RV2009 eine deutliche Verbesserung der Spezifität und Sensitivität beobachtet werden. Im RV2008 hatten nur vier (17,4%) Teilnehmer alle STEC-positiven Proben identifiziert, im RV2009 waren es 14 (53,8%). Die Zahl der falsch-positiven Befunde sank von 17,4% (RV2008) auf 3,8% (RV2009). Die Ermittlung des Konkordanz odds ratio Wertes zeigte signifikante Unterschiede zwischen den teilnehmenden Laboren. Für die Analyse verwendeten die Teilnehmer überwiegend Methoden in Anlehnung an die Amtlichen Untersuchungsverfahren nach § 64 LFGB, wie Stx-ELISA und Kolonie-Immunoblot, oder stx-PCR und Kolonie-DNA-Hybridisierung. Die auffällig niedrige Sensitivität bei Einsatz von Stx-ELISA im RV2008 konnte auf ein kommerzielles Produkt (Novitek Veterinär), das einige Varianten von Stx1 und Stx2 nicht erkannte, zurückgeführt werden. Es wird empfohlen, nur von unabhängiger Seite evaluierte Nachweisverfahren einzusetzen, um die Spezifität und Sensitivität des Testverfahrens zu gewährleisten. Real-Time stx-PCR als neue Methode wurde verstärkt eingesetzt und erwies sich als viel versprechend. Der Nachweis von enterohämorrhagischen E. coli (EHEC) O157:[H7] als häufigster Erreger von Hämorrhagischer Colitis (HC) und Hämolytisch Urämischem Syndrom (HUS) sollte durch Einführung spezifischer Verfahren (ISO16654) verbessert werden, da nur zwei von 26 Teilnehmern diesen Keim aus einer Hackfleischprobe, die zwei STEC Stämme enthielt, isoliert hatten.AbstractFor quality assurance the National Reference Laboratory for Escherichia coli at the Federal Institute for Risk Assessment had organized two ring trials in 2008 and 2009 (called RV2008 and RV2009) on the detection and isolation of Shiga toxin producing Escherichia coli (STEC) from minced beef samples. There were 23 (RV2008) and 26 (RV2009) participants from institutions dealing with the microbiological control of food. For the analysis, the participants received five samples of minced meat containing STEC and four samples without STEC. By comparing the results, a significant improvement of detection sensitivity and specificity was observed for RV2009. In RV2008, only 4 (17.4%) of the participants had identified all STEC-positive samples, compared to 14 (53.8%) in RV2009. The number of false-positive findings decreased from 17.4% (RV2008) to 3.8% (RV2009). Statistically significant differences between the participating laboratories were found by determination of concordance odds ratios. Most participants used methods for detection and isolation of STEC based on protocols that are officially recommended in Germany (§64 LFGB), such as Shiga toxin (Stx) ELISA/Colony Immunoblot, or stx-PCR/DNA colony hybridization. A strikingly low sensitivity when using the Stx-ELISA as detection method was observed for RV2008. This finding could be attributed to the use of a commercial Stx-ELISA (Novitek Veterinär) that showed deficiencies for identification of Stx1 and some variants of Stx2. It is recommended to use only detection systems that have been independently evaluated for their specificity and sensitivity. Real-time stx-PCR as a new method has been increasingly used and proved to be promising. The detection of enterohaemorrhagic E. coli O157: [H7] as the most common cause of Haemorrhagic Colitis (HC) and Haemolytic Uraemic Syndrome (HUS) should be improved by introduction of specific procedures (ISO16654), since only 2 of 26 participants successfully isolated this agent from a minced meat sample that contained two STEC strains.