Zugl.: Giessen, Universiẗat, Diss., 2009.

Universiẗat, Diss., 2007--Giessen.

Zugl.: Giessen, Univ., Diss., 2009

Apesar de Escherichia coli O157:H7 ainda ser considerado o principal sorotipo envolvido com surtos de enfermidades veiculadas por alimentos entre as E. coli produtoras de toxina de Shiga (STEC), outros sorogrupos estão ganhando importância, como O26, O45, O103, O111, O121 e O145, que estão sendo denominados de \"Top Six STEC non O157\". As STEC são responsáveis por sintomas que variam de uma simples diarreia até diarreia sanguinolenta, que pode evoluir ainda para síndrome hemolítica urêmica e púrpura trombótica trombocitopênica, podendo ocasionar danos crônicos como falência renal e levar a óbito. Para tanto, apresentam diversos fatores de virulência, entre eles, as toxinas de Shiga (Stx) ou verotoxinas (Vtx). Os veículos destes micro-organismos são diversos alimentos, sendo o principal deles, as carnes moídas. Apesar da importância da carne moída como veículo transmissor de STEC, pouco se conhece sobre a sua presença nesse alimento comercializado na cidade de São Paulo, SP. Sendo assim, o objetivo deste trabalho foi pesquisar a presença de STEC em carne moída comercializada no varejo da cidade de São Paulo e caracterizar tais isolados quanto à presença dos seguintes fatores de virulência: stx1, stx2, eae e ehx. Foram coletadas 248 amostras em diferentes bairros da cidade de São Paulo. Para a detecção de E. coli sorogrupo O157 foi utilizada a metodologia ISO 16654 e para a detecção dos sorogrupos O103, O111, O145 e O26 foi empregada a metodologia descrita pelo Surveillance Group for Diseases and Infections of Animals (NRM 006). Uma amostra de carne moída (0,4%) apresentou o micro-organismo pesquisado. A identificação genotípica e bioquímica caracterizou esse isolado como STEC O157:H7, portador de todos os fatores de virulência pesquisados: stx1, stx2, eae e ehx. Foi constatada, também, a expressão das proteínas stx em células Vero. Esse é o primeiro relato da presença de E. coli O157:H7 produtora de toxina de Shiga em carne moída no Brasil. / Although Escherichia coli O157:H7 is still considered the most important serotype involved in foodborne disease outbreaks among Shiga toxin-producing E. coli (STEC), other serogroups are receiving more attention such as O26, O45, O103, O111, O121 and O145, that are being called the \"Top Six STEC non O157\". STEC are responsible for symptoms ranging from simple diarrhea to bloody diarrhea, which can further evolve to hemolytic uremic syndrome and thrombotic thrombocytopenic purpura, which may cause damage such as chronic renal failure and lead to death. To do so, they have several virulence factors, including the Shiga toxins (Stx) or verotoxins (Vtx). The vehicles of these microorganisms are many foods, most notably, the ground beef. Despite the importance of ground beef as a vehicle for transmitting STEC, little is known about their presence in this kind of food sold in São Paulo, SP. Therefore, the objective of this study was to investigate the presence of STEC in ground beef sold at retail level in Sao Paulo city and characterize the isolates for the presence of the following virulence factors: stx1, stx2, eae and ehx. 248 samples were collected in different districts of Sao Paulo city. For the detection of E. coli O157 serogroup the methodology ISO 16654 was used and for the detection of serogroups O103, O111, O145 and O26 the methodology described by the Surveillance Group for Diseases and Infections of Animals (NRM 006) was used. One sample of ground beef (0.4%) showed the presence of the microorganism studied. The biochemical and genotypical identification characterized this isolate as STEC O157:H7, carrying all of the investigated virulence factors: stx1, stx2, eae and ehx. The expression of stx proteins in Vero cells was also observed. This is the first report on the isolation of E. coli O157:H7 Shiga toxin-producing from ground beef in Brazil.

Apesar de Escherichia coli O157:H7 ainda ser considerado o principal sorotipo envolvido com surtos de enfermidades veiculadas por alimentos entre as E. coli produtoras de toxina de Shiga (STEC), outros sorogrupos estão ganhando importância, como O26, O45, O103, O111, O121 e O145, que estão sendo denominados de \"Top Six STEC non O157\". As STEC são responsáveis por sintomas que variam de uma simples diarreia até diarreia sanguinolenta, que pode evoluir ainda para síndrome hemolítica urêmica e púrpura trombótica trombocitopênica, podendo ocasionar danos crônicos como falência renal e levar a óbito. Para tanto, apresentam diversos fatores de virulência, entre eles, as toxinas de Shiga (Stx) ou verotoxinas (Vtx). Os veículos destes micro-organismos são diversos alimentos, sendo o principal deles, as carnes moídas. Apesar da importância da carne moída como veículo transmissor de STEC, pouco se conhece sobre a sua presença nesse alimento comercializado na cidade de São Paulo, SP. Sendo assim, o objetivo deste trabalho foi pesquisar a presença de STEC em carne moída comercializada no varejo da cidade de São Paulo e caracterizar tais isolados quanto à presença dos seguintes fatores de virulência: stx1, stx2, eae e ehx. Foram coletadas 248 amostras em diferentes bairros da cidade de São Paulo. Para a detecção de E. coli sorogrupo O157 foi utilizada a metodologia ISO 16654 e para a detecção dos sorogrupos O103, O111, O145 e O26 foi empregada a metodologia descrita pelo Surveillance Group for Diseases and Infections of Animals (NRM 006). Uma amostra de carne moída (0,4%) apresentou o micro-organismo pesquisado. A identificação genotípica e bioquímica caracterizou esse isolado como STEC O157:H7, portador de todos os fatores de virulência pesquisados: stx1, stx2, eae e ehx. Foi constatada, também, a expressão das proteínas stx em células Vero. Esse é o primeiro relato da presença de E. coli O157:H7 produtora de toxina de Shiga em carne moída no Brasil. / Although Escherichia coli O157:H7 is still considered the most important serotype involved in foodborne disease outbreaks among Shiga toxin-producing E. coli (STEC), other serogroups are receiving more attention such as O26, O45, O103, O111, O121 and O145, that are being called the \"Top Six STEC non O157\". STEC are responsible for symptoms ranging from simple diarrhea to bloody diarrhea, which can further evolve to hemolytic uremic syndrome and thrombotic thrombocytopenic purpura, which may cause damage such as chronic renal failure and lead to death. To do so, they have several virulence factors, including the Shiga toxins (Stx) or verotoxins (Vtx). The vehicles of these microorganisms are many foods, most notably, the ground beef. Despite the importance of ground beef as a vehicle for transmitting STEC, little is known about their presence in this kind of food sold in São Paulo, SP. Therefore, the objective of this study was to investigate the presence of STEC in ground beef sold at retail level in Sao Paulo city and characterize the isolates for the presence of the following virulence factors: stx1, stx2, eae and ehx. 248 samples were collected in different districts of Sao Paulo city. For the detection of E. coli O157 serogroup the methodology ISO 16654 was used and for the detection of serogroups O103, O111, O145 and O26 the methodology described by the Surveillance Group for Diseases and Infections of Animals (NRM 006) was used. One sample of ground beef (0.4%) showed the presence of the microorganism studied. The biochemical and genotypical identification characterized this isolate as STEC O157:H7, carrying all of the investigated virulence factors: stx1, stx2, eae and ehx. The expression of stx proteins in Vero cells was also observed. This is the first report on the isolation of E. coli O157:H7 Shiga toxin-producing from ground beef in Brazil.

Sorbitol fermentierende (SF) Shiga Toxin-Produzierende Escherichia coli (STEC)-Stämme des Serotyps O157:H- stellen bedeutsame Auslöser für Durchfallerkrankungen und des lebensbedrohlichen Hämolytisch-Urämischen Syndroms (HUS) in Mitteleuropa dar. Die Virulenzfaktoren und Krankheitsbilder solcher Stämme ähneln denen von STEC O157:H7-Stämmen, welche weltweit am häufigsten innerhalb der O157-Serogruppe isoliert werden. Für zwei STEC O157:H7-Stämme ist die komplette Genomsequenz bereits veröffentlicht. Über SF STEC O157:H- sind hingegen nur einige genetische Unterschiede mit STEC O157:H7 bezüglich der Ausstattung an Virulenzfaktoren bekannt. Diese sind vorwiegend auf den Plasmiden beider Serotypen kodiert. In einem Modell, das die Entstehungsweise des O157-Komplexes zu erklären versucht, wird der nicht begeißelte, Sorbit fermentierende O157:H- -Serotyp als eigene phylogenetische Linie angesehen. Um einen tieferen Einblick in die genomische Diversität dieser beiden O157-Linien zu erhalten, wurden verschiedene molekularbiologische Methoden eingesetzt. Als Modellstamm für SF STEC O157:H- diente der Stamm 493/89, welcher von einem HUS-Patienten aus Deutschland isoliert wurde. Zum einen wurde eine Cosmid-Genbank des Stamms 493/89 sequenziert und mit dem STEC O157:H7-Referenzstamm EDL933 verglichen. Des weiteren wurde eine subtraktive suppressive Hybridisierung (SSH) mit beiden Stämmen des gleichen Serotyps durchgeführt. Ein Vergleich beider Genome mit dem kompletten Genom des apathogenen Laborstammes E. coli K-12 erfolgte über die Makroarraytechnik. Schließlich wurde der Stamm 493/89 mit Hilfe eines Makroarrays, auf dem bekannte Virulenz-assoziierte DNA-Sequenzen untergebracht sind (Pathoarray) untersucht. Nach Anwendung dieser Techniken konnten zwei weitere Gene in SF STEC O157:H- identifiziert werden, die für potentielle Virulenzfaktoren kodieren. Mit dem Gen für den ?EHEC factor for adherence? (Efa1), welcher gleichzeitig auch als Lymphostatin (LifA) beschrieben wird, ist in SF O157:H- ein multifunktionelles Gen vorhanden, welches nur fragmentiert in O157:H7 präsent ist. In 90 % der getesteten SF O157:H- -Stämme wurden außerdem die Gene für das ?Cytolethal Distending Toxin? (CDT) detektiert. Diese weisen die größte Ähnlichkeit zu cdt-III des E. coli O15:H21-Stammes S5 auf, welche dort auf einem Plasmid kodiert vorliegen. Im Gegensatz dazu sprechen die flankierenden Sequenzen von cdt in E. coli 493/89 für ein Gen, das durch einen temperenten Phagen aufgenommen wurde. So konnte cdt, wenn auch nur in geringem Ausmaß in STEC O157:H7-Stämmen durch PCR-Analyse gefunden werden. Die beiden Makroarrays lieferten für beide Serovare eine bemerkenswerte Anzahl spezifischer Sequenzen. Der E. coli K-12 Makroarray bekräftigt zudem die größere Ähnlichkeit des nicht pathogenen E. coli K-12 Genoms mit SF STEC O157:H- als mit STEC O157:H7. Mit Hilfe der Cosmid-Genbank wurde in SF STEC O157:H- eine 8,8 Kb große Sequenz ermittelt, die phagenhomologe Bestandteile der Shigella-Resistance Locus (SRL)-Pathogenitätsinsel (PAI) von Shigella flexneri 2a enthält. Diese Sequenz fehlt in STEC O157:H7 und deutet Gemeinsamkeiten für SF STEC O157:H- und Shigella flexneri 2a bezüglich ihrer Phagentransduktion an. Die Präsenz von efa1/lifA, cdt und der 8,8 Kb großen SRL-homologen Sequenz wurde auch bei E. coli O55:H- -Stämmen durch PCR überprüft. Diese werden als vermutliche Vorstufe des O157-Komplexes angesehen. Die Ergebnisse lassen erkennen, dass efa1/lifA bereits in diesem E. coli O55-Vorläufer vorhanden war, die SRL-homologe Sequenz aber erst vor und cdt nach der Abzweigung in die O157:H- -Linie erhalten wurde. In O157:H7 wurden efa1/lifA und der SRL-homologe Genbereich teilweise oder vollständig deletiert. Die Ergebnisse bestätigen in ihrer Gesamtheit SF STEC O157:H- als ein eigenständig entwickeltes Pathogen mit klar erkennbaren Unterschieden zu STEC O157:H7 und bekräftigen damit seine bedeutsame Position innerhalb der epidemiologisch relevanten STEC-Serogruppen. / Sorbitol-fermenting (SF) Shiga toxin-producing Escherichia coli (STEC) strains of serotype O157:H- (nonmotile) are important causes of diarrhea and the life-threatening hemolytic uremic syndrome (HUS) in Europe. The virulence characteristics of these strains are similar to those of STEC O157:H7, which is the most frequently isolated STEC serotype causing human diseases worldwide. Recently, the complete genome sequences of two STEC O157:H7 strains have been published. In contrast, the sequences of SF STEC O157:H- remain largely uncharacterized. Therefore, differences in virulence characteristics of the two O157 pathogens are poorly understood and have been till now limited to genes encoded on a large plasmid. In a stepwise evolutionary model proposed for the STEC O157 complex, SF STEC O157:H- strains are classified as a unique phylogenetic lineage. In this work, we used different molecular approaches to gain more insight into the genomic diversity of the two O157 lineages. First, a cosmid gene library of a SF STEC O157:H- strain 493/89, which was isolated from an HUS patient in Germany, was sequenced and compared to the genomic sequences of the STEC O157:H7 reference strain EDL933. Second, a subtractive suppressive hybridization (SSH) between these two strains was performed. Third, the genomes of both O157 pathogens were compared to that of E. coli strain K-12 using a macroarray. Finally, the SF STEC O157:H- strain 493/89 was analyzed for DNA sequences of major virulence genes using a pathoarray. Using these techniques, two genes encoding potential virulence factors, which are specific for SF STEC O157:H-, have been identified. These include i) a 9669-bp open reading frame (ORF) encoding a multifunctional gene efa1/lifA, which is only rudimentarily present in STEC O157:H7; ii) the gene cluster enoding the cytolethal distending toxin (CDT). The cdt cluster of SF STEC O157:H- demonstrates a high similarity to cdt-III of E. coli O15:H21 strain S5, which was reported to be plasmid-encoded. In contrast, the flanking regions of cdt in strain 493/89 indicate the acquisition of cdt via a temperate bacteriophage. In accordance with this hypothesis, cdt could be detected, using a PCR strategy, in 90% of SF STEC O157:H-, but also in STEC O157:H7 strains, even though with a low frequency. Both macroarray methods identified a remarkable amount of specific differences between both O157 serotypes. Moreover, the E. coli K-12 macroarray confirmed a higher similarity of nonpathogenic E. coli K-12 to SF STEC O157:H- than to STEC O157:H7. By sequencing the cosmid library, an 8,8 kb long sequence could be detected in SF STEC O157:H-, which contains elements of the Shigella resistance locus (SRL) pathogenicity island (PAI). These elements are also homologous to bacteriophage sequences. This 8,8 kb sequence is missing in STEC O157:H7 and suggests a common acquisition of bacteriophage elements in SF STEC O157:H- and Shigella flexneri 2a. The presence of efa1/lifA, cdt and the 8,8 kb SRL-homologue sequence was also tested by PCR in E. coli O55:H7 strains which are proposed to be ancestors of the O157 complex. The results indicate, that efa1/lifA was already present in the O157 precursor, but the SRL-homologue sequence was received shortly before branching off the O157:H- lineage. Both sequences were completely or partially deleted in O157:H7. Based on the PCR results, cdt was acquired by the SF STEC O157:H- serotype after this lineage diverged from E. coli O157:H7. In conclusion, all results confirm that SF STEC O157:H- represent a distinct pathogenic group of STEC O157 which demonstrates remarkable genomic, virulence and evolutionary differences from STEC O157:H7.

Shiga Toxin-produzierende Escherichia coli (STEC) verursachen Diarrhöen und hämorrhagische Colitis. Als lebensbedrohliche Komplikation können sie ein hämolytisch-urämisches Syndrom auslösen. Bisher identifizierte Virulenz-faktoren der STEC sind auf Bakteriophagen, Plasmiden und Pathogenitätsinseln kodiert. Bei der Mehrzahl der klinischen STEC-Isolate konnte die Pathogen-itätsinsel LEE (locus of enterocyte effacement) nachgewiesen werden. Der LEE ist stromabwärts des Gens für eine Selenocystein-tRNA (selC) ins Chromosom integriert. Ziel der vorliegenden Arbeit war die Charakterisierung von STEC, denen der LEE fehlt und die funktionelle Analyse von potentiellen Virulenz-assoziierten Genen. Dabei wurde eine Fokussierung auf Gene vorgenommen, die im Bereich der selC-Region vorkommen. Mittels PCR wurde zunächst überprüft, ob bei den LEE-negativen STEC-Stämmen Fremd-DNA in der selC-Region integriert ist. Hierzu wurden 35 LEE-negative STEC getestet. Bei 13 Stämmen konnte eine Insertion von Fremd-DNA gezeigt werden. Von einem dieser Stämme (E. coli 4797/97, Serovar O91:H-) wurde aus einer Genbank ein die selC-Region enthaltendes DNA-Fragment identifiziert. Ein 37,7 kb großes Fragment dieses Cosmids wurde vollständig sequenziert. Die Analyse der gesamten Sequenz ergab 30 offene Leserahmen mit Längen zwischen 95 und 4092 bp. Der G+C-Gehalt betrug 47,4%. An mehreren Stellen wurden Bereiche mit hoher Homologie zu verschiedenen Insertionssequenzen, inverted repeats und Integrasen gefunden. Drei Leserahmen hatten eine hohe Homologie zu bereits bekannten Genen. Hierbei handelt es sich um die iha- , btuB- und espP-Gene von E. coli. Das iha-Gen kodiert für ein Adhärenz-vermittelndes Protein, btuB für den Vitamin B12 Rezeptor und espP für eine Serinprotease. Bei den iha-Adhäsinen und Serinproteasen handelt es sich um potentielle Pathogenitätsfaktoren. Der sequenzierte Bereich hat somit die charakteristischen Eigenschaften einer Pathogenitätsinsel: Die Präsenz von mehreren Pathogenitätsgenen und mobilen Elementen (Insertionselemente, Integrasen), die Lokalisation nahe an tRNA-Genen sowie ein veränderter G+C-Gehalt gegenüber dem Restgenom. Zur weiteren Charakterisierung des espI-Genprodukts wurde espI subkloniert. Bei der Untersuchung von Kulturüberständen zeigte sich, dass der Subklon DH5/pzh4 ein Protein von etwa 110 kDa sezernierte. In weiteren Experimenten konnte gezeigt werden, dass die Serinprotease des Stammes 4797/97 als 140,8 kDa großes Vorläuferprotein synthetisiert und während des anschließenden Exportvorganges sowohl N-terminal als auch C-terminal prozessiert wird. Das C-terminale Ende wirkt als Translokator durch die äußere Membran, wo es nach Abspaltung des reifen EspI-Proteins verbleibt. Das reife Protein weist eine Größe von 110,5 kDa auf. Der hier gezeigte Transportmechanismus ist charakteristisch für sogenannte Autotransporter-Proteine. Trotz der hohen Sequenzhomologie zur IgA1-Protease von Neisseria gonorrhoeae, die als Prototyp der Autotransporter-Proteine gilt, konnte für EspI keine Proteaseaktivität gegenüber IgA gezeigt werden. EspI weist auch hohe Sequenzhomologie zu Exoproteinen von Shigella flexneri (SepA, Mucinase und SigA), EHEC (EspP) und vogelpathogenen E. coli (Tsh) auf, allerdings konnte keines der Substrate dieser Proteasen von EspI gespalten werden. Dagegen konnte eine proteolytische Aktivität gegenüber Schweine-Pepsin A und Apolipoprotein A1 nachgewiesen werden. Die putative Virulenz und räumliche Nähe der Gene espI, btuB und iha macht diese Region zum interessantesten Stück der im Rahmen dieser Arbeit identifizierten neuen Pathogenitätsinsel. Eine PCR-Untersuchung zur Verbreitung dieser Gene zeigte, dass diese Region bei LEE-negativen STEC weit verbreitet ist. Inwieweit diese Pathogenitätsinsel einen Einfluss auf die Virulenz von STEC hat, muss in weiteren Experimenten geklärt werden. / Shiga toxin-producing Escherichia coli (STEC) cause diarrhea and hemorrhagic colitis. As life-threatening disease they can trigger a hemolytic-uremic syndrome. Virulence factors of STEC identified so far are encoded on bacterio-phages, plasmids and pathogenicity islands. The majority of clinical STEC-isolates could be proven to harbour the pathogenicity island LEE (locus of enterocyte effacement). LEE is integrated in the chromosome downstream of the gene encoding a selenocystein-tRNA (selC). Target of this work was the characterisation of STEC lacking LEE and the functional analysis of potential virulence-associated genes. The focus was put on genes found in the selC-region. Using PCR analysis it was checked whether LEE-negative STEC-strains have integrated foreign DNA. Of 35 LEE-negative STEC 13 strains could be shown to have an insertion of foreign DNA. From one of these strains (E.coli 4797/97, serogroup O91:H-) a DNA-fragment was detected from a cosmid library that harboured the selC region. The sequence of a 37,7 kb long fragment of this cosmid was fully analysed. 30 open reading frames (ORF) between 95 and 4092 bp length were identified. The G+C-content was 47,4 %. In different parts the sequence shows a high homology to diverse insertion sequences, inverted repeats and integrase genes. Three ORF had high homology to already known genes, which are the iha- btuB- and espP-genes of E.coli. The iha-gene is encoding an adherence-conferring protein, btuB the receptor for vitamin B12 and espP a serine protease. Iha-adhesines and serine proteases are potential pathogenicity factors. Thus the sequenced area shows the character-istic attributes of a pathogenicity island: The presence of different pathogenicity genes and mobile elements (insertion elements, integrases), the localisation next to a tRNA-gene and a G+C-content different from that of the whole genome.We termed the espP-homologue espI for "extracellular serine protease encoded on an island". For further characterisation of its product, espI was subcloned. In the analysis of culture supernatants its size was about 110 kDa. In following experiments it could be proven that the serine protease of the strain 4797/97 is synthesised as a 140,8 kDa protein and cleaved on the N- and C-terminal part while its export through the cell membrane. The C-terminal part functions as a locater through the outer membrane, where it remains after the cleavage of the mature EspI-protein. The size of this mature protein is 110,5 kDa. The transport mechanism found here is characteristic for so called autotransporter-proteins. Despite the high sequence-homology to the IgA1-protease of Neisseria gonorrhoae, which is taken as prototype of autotransporter proteins, a proteolytic activity of EspI towards IgA could not be shown. There are also high sequence-homologies to exoproteins of Shigella flexneri (SepA, mucinase and SigA), EHEC (EspP) and avian pathogenic E.coli (Tsh). Nevertheless none of the substrates of these proteases could be cleaved by EspI. In contrast a pretolytic activity against swine pepsine A1 and apolipoprotein A1 could be shown.The putative virulence and the close location of the genes espI, btuB and iha make this region the most interesting part of the new pathogenicity island identified in this work. A PCR-analysis about the spreading of these genes showed, that this region is widespread amongst LEE-negative STEC. The influence of this pathogenicity island on the virulence of STEC has to be clarified in further experiments.

Würzburg, Univ., Diss., 2002.

BACKGROUND: In the U.S. STEC HUS is the most common cause of acute renal failure in children. In TN from 1996-2017 there were 2008 STEC cases were reported. Every year in the U.S, there 36 reported mortality each year. At our local children’s hospital, 4-5 children are hospitalized with STEC infection each year. Some of these children had no history of ingesting food items that could have placed them at risk to develop STEC infection; however, there are other ways that humans could get infected, such as exposure to contaminated water from cattle farms. GOALS: To determine if there are differences in the presence of STEC at a local park at different seasons of the year. METHODS: Fifty (50) ml of water samples were collected from a creek in 2 areas of public park in Johnson City, TN. Samples were inoculated to Sorbitol McConkey Agar (SMAC) plates under sterile techniques & incubated at 36C for 18 hours under aerobic conditions. RESULTS: Table demonstrating presence of STEC from water samples at different seasons of the year. SEASON OF THE YEAR # COLONIES FOUNDERS PARK # COLONIES LIBRARY PARK SUMMER JUNE 2018 A:1 B:1 C:2 TOTAL: 4 A: 3 B: 2 C: 1 TOTAL: 6 FALL SEPT 2018 A: 1 B: 3 C: 2 TOTAL: 6 A: 1 B:2 C:4 TOTAL: 7 WINTER DEC 2018 A: 1 B: 0 C: 1 TOTAL: 2 A: 0 B: 1 C: 1 TOTAL: 2 SPRING MARCH 2019 A: 2 B: 2 C:1 TOTAL: 5 A: 0 B: 0 C: 0 TOTAL: 0 DISCUSSION/ CONCLUSION: STEC was present at almost every season of the year. Public health measures should be undertaken to inform the community that these waters around public parks are contaminated with STEC to prevent STEC infection. References: TN Dept of Health CEDEP report CDC website

Master of Science / Department of Food Science / Randall Phebus / Shiga toxin-producing Escherichia coli (STEC) are Gram negative rod-shaped bacteria that are causative agents of foodborne disease. While natural flora in the gastrointestinal tracts of bovines and other ruminants, certain enterohemorrhagic STEC strains cause serious or even fatal disease when ingested. In 2012, the USDA identified six STEC serotypes (O26, O45, O103, O111, O121, O145) as particularly dangerous (in addition to O157:H7; STEC-7) based upon data from the Centers for Disease Control and Prevention and designated them adulterants in raw ground beef, its component materials, and non-intact raw beef products. This research addressed three important components for detection of the recently declared STEC adulterant serotypes. Part one evaluated traditional E. coli O157 selective enrichment media, and additional media types and additive levels, for STEC-7 cultural amplification. Buffered peptone water (BPW), universal pre-enrichment broth (UPB), tryptic soy broth (TSB), TSB with 8 mg/L novobiocin, Escherichia coli broth (EC), and EC with 5, 8, and 20 mg/L novobiocin added were evaluated. EC broth performed equally well compared to non-selective media types, but addition of novobiocin supplement, typically used to suppress overgrowth by background flora, suppressed non-O157 STEC growth. Higher levels of novobiocin inhibited most serotypes. Part two tested the ability of transport media to maintain original STEC-7 concentrations as samples are transported to analytical laboratories. Transport media BPW, maximum recovery diluent (MRD), and Cary-Blair transport medium (CB) were inoculated with individual STEC serotypes and held at 4 or 10 °C for 72 h. Growth/survival curves indicated that CB maintained STEC-7 populations nearest to inoculation levels during storage at either temperature. Part three, part of a field study determining STEC-7 prevalence rates for cattle, hides and dressed carcasses, compared qualitative results generated by two molecular-based detection systems (BioControl Assurance GDS® and Neogen NeoSeek™), analyzing 576 selectively enriched beef carcass sponge samples collected from a commercial processing facility. The GDS and NeoSeek systems indicated 28.7 and 6.1 percent presumptive positive rates for STEC-7, respectively. It was speculated the higher GDS prevalence rate was due to false-positive determinations from the mixed culture enrichments, as viable STEC-7 cells were not recovered by immuno-magnetic bead culture isolation.