O consumo de vegetais e de frutas tem aumentado mundialmente, bem como os surtos alimentares envolvendo esses alimentos, especialmente a alface que é o vegetal folhoso mais consumido em nível mundial. Dessa forma, o objetivo desse estudo foi realizar uma avaliação quantitativa do risco de infecções causadas por Salmonella spp. e por Escherichia coli O157:H7 a partir do consumo de alface produzida e consumida no Rio Grande do Sul, visto que esses patógenos são os mais relacionados a surtos alimentares envolvendo vegetais folhosos em nível mundial. Para melhor compreender o comportamento desses patógenos na alface, eles foram inoculados nesse vegetal separadamente e armazenados sob condições isotérmicas de 5 a 40°C para Salmonella e de 5 a 42ºC para E. coli O157:H7, bem como sob condições não isotérmicas, simulando temperaturas encontradas da colheita até a venda da alface no Rio Grande do Sul. Dados experimentais demonstraram que ambas as bactérias podem se multiplicar em todas as temperaturas examinadas. Também foi proposto um parâmetro de tempo de multiplicação insignificante (ς), o qual fornece o tempo em que a alface pode ser exposta a uma temperatura específica e não apresentar uma multiplicação expressiva. O ς foi desenvolvido com base na equação do modelo primário de Baranyi e no conceito do potencial de crescimento. ς é o valor da fase lag adicionado do tempo necessário para população microbiana aumentar 0,5 log UFC/g. O ς da alface exposta a 37 °C foi de 1,3 h, enquanto que a 5 °C foi de 3,3 dias. Além dos modelos adequados, dados de prevalência e concentração são primordiais na avaliação de risco. Assim, foi realizada uma revisão sistemática da literatura para buscar esses dados A prevalência mundial encontrada foi de 0,041 para ambos os patógenos na alface. Já a prevalência dos países desenvolvidos foi de 0,028 para Salmonella e de 0,125 para E. coli (EHEC), enquanto que nos países em desenvolvimento foi de 0,064 para Salmonella e 0,024 para E. coli (EHEC). A concentração de Salmonella em alface, em países em desenvolvimento, variou de 4,57 a 218,78 NMP/g, e para E. coli (EHEC) a concentração foi de < 3,0 NMP/g até > 1100 NMP/g. O modelo de avaliação quantitativa de risco microbiológico foi composto por nove módulos, desde o armazenamento da alface nas fazendas produtoras até o consumo. O risco médio (baseado no cenário mais comumente encontrado no Rio Grande do Sul) de infecção por Salmonella por mês foi de 0,017, enquanto que por E. coli O157:H7 foi de 0,006. Assim, de modo geral, o risco de infecção por Salmonella é maior do que por E. coli O157:H7 quando a alface é produzida e consumida nesse estado. Todos os cenários alternativos à correta higienização da alface (lavar as folhas de alface com água potável seguido de imersão em 200 ppm de cloro livre, por 15 minutos e enxaguar com água potável) aumentaram o risco. A principal redução do risco foi identificada no cenário que considerou o uso de refrigeração em todos os módulos do modelo. Análises de sensibilidade indicaram que, além da manutenção da cadeia fria e do procedimento correto de higienização, é importante reduzir a prevalência e a concentração dos patógenos na alface, a fim de diminuir o risco de infecção por essas bactérias. Por fim, a avaliação de risco desenvolvida nessa tese pode auxiliar no desenvolvimento de estratégias de intervenção para mitigar esse risco. / The consumption of vegetables and fruits has increased worldwide, as well as foodborne outbreaks involving these foods, especially lettuce that is the most consumed leafy vegetable in the world. Thus, the objective of this study was to carry out a quantitative microbial risk assessment of Salmonella spp. and Escherichia coli O157: H7 on lettuce produced and consumed in Rio Grande do Sul, since these pathogens are the most related to foodborne outbreaks involving leafy vegetables worldwide. To study the behavior of these pathogens on lettuce, they were inoculated on this vegetable separately and stored under isothermal conditions of 5 to 40 °C for Salmonella and 5 to 42 °C for E. coli O157:H7, as well as under non-isothermal conditions, simulating temperatures from the harvest until the sale of lettuce in Rio Grande do Sul. Experimental data demonstrated that both bacteria can growth at all temperatures examined. A negligible growth time parameter (ς) has also been proposed, which provides the time that lettuce can be exposed to a specific temperature and does not present an expressive growth. The ς was developed based on the equation of the Baranyi primary model and the concept of growth potential. ς is the lag phase added value of the time required for microbial population to increase 0.5 log CFU/g. The ς of lettuce exposed at 37 ºC was 1.3 h, whereas at 5 ºC it was 3.3 days. In addition, prevalence and concentration data are paramount in the risk assessment studies. Thus, a systematic review of the literature was carried out to collect these data. The global prevalence found was 0.041 for both pathogens in lettuce The prevalence of developed countries was 0.028 for Salmonella and 0.125 for E. coli (EHEC), while in developing countries it was 0.064 for Salmonella and 0.024 for E. coli (EHEC). The concentration of Salmonella in lettuce in developing countries ranged from 4.57 to 218.78 MPN/g, and for E. coli (EHEC) the concentration was < 3.0 MPN/g to > 1100 MPN/g. The quantitative microbial risk assessment model was composed by nine modules, from lettuce storage on farms to consumption. The average risk (based on the scenario most commonly found in Rio Grande do Sul) of Salmonella infection per month was 0.017, whereas for E. coli O157:H7 it was 0.006. Thus, in general, the risk of infection by Salmonella is higher than by E. coli O157:H7 when lettuce is produced and consumed in this State. All scenarios that were alternative to the correct hygiene of lettuce (washing lettuce leaves with drinking water followed by immersion in 200 ppm of free chlorine for 15 minutes and rinsing with potable water) increased the risk. The main risk reduction was identified in the scenario that considered the use of refrigeration in all modules of the model. Sensitivity analyzes indicated that, in addition to maintaining the cold chain and the correct hygienization procedure, it is important to reduce the prevalence and concentration of pathogens in lettuce, in order to reduce the risk of infection by these bacteria. Finally, the risk assessment developed in this thesis can help in the development of intervention strategies to mitigate this risk.

Cells defend their cytosol against pathogen invasion using cell-autonomous immunity. When pathogens enter the cytosol they can damage host endomembranes, causing the mislocalisation of host molecules not normally found in the cytosol that are sensed as Danger Associated Molecular Patterns (DAMPs). Glycans exposed on damaged endomembranes are detected by danger receptors such as Galectin8. Galectin8 is recognised by the autophagy cargo receptor NDP52, specifically targeting the bacteria to autophagy. I hypothesised that other proteins would also be recruited to damaged endomembranes, which may initiate downstream mechanisms involved in cell-autonomous immunity or endomembrane repair. Identifying novel damage recruited proteins (DRPs) is difficult due to the short-lived and dynamic nature of damaged endomembranes. Therefore, I developed an unbiased approach for the identification of novel DRPs by proximity-dependent biotinylation using the ascorbate peroxidise enzyme APEX. This approach preferentially labels proteins located at damaged endomembranes for subsequent identification by TMT mass spectrometry. Four enriched proteins CCDC50, FBXO21, STAMBP and PDCD6 were identified as novel damage recruited proteins, recognising damaged SCVs. An alternative form of cell-autonomous immunity is the induction of cell death, for example by pyroptosis. Cell death destroys the bacteria's replicative niche and exposes them to the extracellular space where they may be phagocytosed. I hypothesised that host cells might tag cytoplasmic bacteria with intracellular opsonins to assist in their phagocytosis following their release from host cells. However, my work revealed that intracellular Salmonella Typhimurium acquire phagocytosis protection, thus becoming internalised by phagocytes less efficiently than control bacteria. Phagocytosis protection was acquired rapidly after S.Typhimurium infection and was not observed with dead bacteria. Phagocytosis protection is only partially reversed by opsonisation in human serum. My results indicate that intracellular S.Typhimurium-induces an evasion mechanism to prevent its subsequent recognition by extracellular phagocytes.

No description available.

Non-coding RNAs constitute a major class of regulators involved in bacterial gene expression. A group of riboregulators of heterogeneous size and shape referred to as small regulatory RNAs (sRNAs) control trans- or cis-encoded genes through direct base-pairing with their mRNAs. Although mostly inhibiting their target mRNAs, several sRNAs also induce gene expression. An important co-factor for sRNA activity is the RNA chaperone, Hfq, which is able to rearrange intramolecular secondary structures and to promote annealing of complementary RNA sequences. In addition, Hfq protects unpaired RNA from degradation by ribonucleases and thus increases sRNA stability. Co-immunoprecipitation of RNA with the Hfq protein, and further experimental as well as bioinformatical studies performed over the last decade suggested the presence of more than 150 different sRNAs in various Enterobacteria including Escherichia coli and Salmonellae. So-called core sRNAs are considered to fulfill central cellular activities as deduced from their high degree of conservation among different species. Approximately 25 core sRNAs have been implicated in gene regulation under a variety of environmental responses. However, for the majority of sRNAs, both the riboregulators’ individual biological roles as well as modes of action remain to be elucidated. The current study aimed to define the cellular functions of the two highly conserved, Hfq-dependent sRNAs, SdsR and RydC, in the model pathogen Salmonella Typhimurium. SdsR had been known as one of the most abundant sRNAs during stationary growth phase in E. coli. Examination of the conservation patterns in the sdsR promoter region in combination with classic genetic analyses revealed SdsR as the first sRNA under direct transcriptional control of the alternative σ factor σS. In Salmonella, over-expression of SdsR down-regulates the synthesis of the major porin OmpD, and the interaction site in the ompD mRNA coding sequence was mapped by a 3'RACE-based approach. At the post-transcriptional level, expression of ompD is controlled by three additional sRNAs, but SdsR plays a specific role in porin regulation during the stringent response. Similarly, RydC, the second sRNA adressed in this study, was initially discovered in E. coli but appeared to be conserved in many related γ-proteobacteria. An interesting aspect of this Hfq-dependent sRNAs is its secondary structure involving a pseudo-knot configuration, while the 5’ end remains single stranded. A transcriptomic approach combining RydC pulse-expression and scoring of global mRNA changes on microarrays was employed to identify the targets of this sRNA. RydC specifically activated expression of the longer of two versions of the cfa mRNA encoding for the phospholipid-modifying enzyme cyclopropane fatty acid synthase. Employing its conserved single-stranded 5' end, RydC acts as a positive regulator and masks a recognition site of the endoribonuclease, RNase E, in the cfa leader. / Die bakterielle Genexpression wird unter anderem maßgeblich von nicht-kodierenden RNAs bestimmt. Kleine regulatorische RNAs (sRNAs) sind eine bezüglich Größe und Struktur heterogene Gruppe von Riboregulatoren, die ihre in cis oder in trans-kodierten Zielgene mittels direkter Basenpaarungen kontrollieren. Während der Großteil der sRNAs reprimierend wirkt, konnte für einige RNAs gezeigt werden, dass sie die Expression ihres Zieltranskripts verstärken. Ein wichtiger Kofaktor für die regulatorische Funktion der sRNAs ist das RNA-Chaperon Hfq, welches sowohl die Umfaltung intramolekularer Sekundärstrukturen ermöglicht, als auch die Ausbildung von Basenpaarungen zwischen komplementären RNA-Sequenzen steuert. Zusätzlich schützt Hfq nicht-gepaarte RNAs vor dem Abbau durch Ribonukleasen, und trägt damit zur Stabilität der Moleküle bei. Durch Ko-Immunopräzipitation mit Hfq sowie in weiteren experimentellen als auch bioinformatischen Studien konnten im letzten Jahrzehnt in diversen Enterobakterien, wie z.B. auch Escherichia coli und Salmonellae, mehr als 150 verschiedene sRNAs bestimmt werden. Von so genannten "core sRNAs" (Kern-sRNAs) wird aufgrund ihres hohen Grades an Konservierung in unterschiedlichen Spezies angenommen, dass sie zentrale Funktionen erfüllen. Etwa 25 core sRNAs agieren unter verschiedenen Umweltbedingungen als Regulatoren. Ihre exakte biologische Rolle, sowie ihre Funktionsweise sind jedoch größtenteils noch unbekannt. In der vorliegenden Arbeit wurden die beiden konservierten, Hfq-abhängigen sRNAs, SdsR und RydC, im Modellpathogen Salmonella Typhimurium charakterisiert. SdsR war als eine der abundantesten sRNAs der stationären Phase in E. coli beschrieben worden. Durch Auswertung der Konservierungsmuster der sdsR Promotorsequenz sowie klassische genetische Analyse konnte SdsR als erste sRNA unter direkter Kontrolle des alternativen σ Faktors σS bestimmt werden. In Salmonella führt die Überexpression von SdsR zur Reprimierung des Membranporins OmpD, und die Bindestelle von SdsR auf dem ompD Transkript wurde mittels einer auf 3'-RACE basierenden Methode ermittelt. Obwohl die Expression von ompD auf post-transkriptionaler Ebene von drei weiteren sRNAs kontrolliert wird, konnte eine spezische Regulation des Porins durch SdsR während Aminosäure-Hungerung gezeigt werden. Auch RydC, die zweite in dieser Studie analysierte sRNA, wurde zunächst in E. coli beschrieben und ist aber auch in weiteren γ-Proteobakterien konserviert. Interessanterweise enthält die Sekundärstruktur dieser Hfq-abhängigen sRNA einen Pseudoknoten, während das 5'-Ende ungepaart ist. Die Zielgene von RydC wurden mittels einer Transkriptomanalyse bestimmt, in der die Änderung der Häufigkeitsverteilung aller mRNAs nach kurzzeitiger Überexpression der sRNA auf Microarrays untersucht wurde. RydC bewirkte die spezifische Aktivierung des längeren von insgesamt zwei Versionen der cfa mRNA, die für eine Cyclopropan-fettsäuresynthase kodiert, ein Enzym das zur Modifikation von Phospholipiden dient. Eine Basenpaarung über das freie 5'-Ende der sRNA RydC führt zur Aktivierung der cfa-Expression, und maskiert eine Erkennungssequenz der Endoribonuklease, RNase E, innerhalb des Transkripts.

The infection of a eukaryotic host cell by a bacterial pathogen is one of the most intimate examples of cross-kingdom interactions in biology. Infection processes are highly relevant from both a basic research as well as a clinical point of view. Sophisticated mechanisms have evolved in the pathogen to manipulate the host response and vice versa host cells have developed a wide range of anti-microbial defense strategies to combat bacterial invasion and clear infections. However, it is this diversity and complexity that makes infection research so challenging to technically address as common approaches have either been optimized for bacterial or eukaryotic organisms. Instead, methods are required that are able to deal with the often dramatic discrepancy between host and pathogen with respect to various cellular properties and processes. One class of cellular macromolecules that exemplify this host-pathogen heterogeneity is given by their transcriptomes: Bacterial transcripts differ from their eukaryotic counterparts in many aspects that involve both quantitative and qualitative traits. The entity of RNA transcripts present in a cell is of paramount interest as it reflects the cell’s physiological state under the given condition. Genome-wide transcriptomic techniques such as RNA-seq have therefore been used for single-organism analyses for several years, but their applicability has been limited for infection studies. The present work describes the establishment of a novel transcriptomic approach for infection biology which we have termed “Dual RNA-seq”. Using this technology, it was intended to shed light particularly on the contribution of non-protein-encoding transcripts to virulence, as these classes have mostly evaded previous infection studies due to the lack of suitable methods. The performance of Dual RNA-seq was evaluated in an in vitro infection model based on the important facultative intracellular pathogen Salmonella enterica serovar Typhimurium and different human cell lines. Dual RNA-seq was found to be capable of capturing all major bacterial and human transcript classes and proved reproducible. During the course of these experiments, a previously largely uncharacterized bacterial small non-coding RNA (sRNA), referred to as STnc440, was identified as one of the most strongly induced genes in intracellular Salmonella. Interestingly, while inhibition of STnc440 expression has been previously shown to cause a virulence defect in different animal models of Salmonellosis, the underlying molecular mechanisms have remained obscure. Here, classical genetics, transcriptomics and biochemical assays proposed a complex model of Salmonella gene expression control that is orchestrated by this sRNA. In particular, STnc440 was found to be involved in the regulation of multiple bacterial target mRNAs by direct base pair interaction with consequences for Salmonella virulence and implications for the host’s immune response. These findings exemplify the scope of Dual RNA-seq for the identification and characterization of novel bacterial virulence factors during host infection. / Die Infektion einer eukaryontischen Wirtszelle mit einem bakteriellen Pathogen ist eines der komplexesten Beispiele einer Domänen-überschreitenden Wechselwirkung zweier Organismen. Infektionsprozesse sind in höchstem Maße relevant, sowohl in der Grundforschung als auch von einem klinischen Blickwinkel aus betrachtet. Im Laufe der Evolution entstanden komplizierte Mechanismen, die es einem Pathogen erlauben, die Wirtsantwort zu manipulieren. Umgekehrt haben potentielle Wirtszellen eine Reihe von anti-mikrobiellen Verteidigungsstrategien entwickelt, um bakterielle Infektionen zu bekämpfen und letztlich zu beseitigen. Es sind jedoch genau diese Verschiedenheit und Komplexität, welche die Infektionsforschung so anspruchsvoll und technisch schwer analysierbar machen. Gängige Analysemethoden wurden zumeist entweder für bakterielle oder aber eukaryontische Organismen entwickelt. Dagegen werden Techniken benötigt, welche es erlauben, mit den mitunter extremen Unterschieden zwischen Wirt und Pathogen umzugehen, die sich in etlichen zellulären Eigenschaften und Prozessen manifestieren. Eine Klasse zellulärer Makromoleküle, die diese Heterogenität zwischen Wirt und Pathogen widerspiegelt, sind ihre jeweiligen Transkriptome: Bakterielle Transkripte unter-scheiden sich von ihren eukaryontischen Pendants in vielerlei Hinsicht, was sowohl quantitative als auch qualitative Aspekte miteinschließt. Die Gesamtheit zellulärer Transkripte ist von größter Bedeutung, da sie den physiologischen Zustand der jeweiligen Zelle unter den gegebenen Bedingungen reflektiert. Aus diesem Grund werden Genom-weite Transkriptom-techniken wie etwa die RNA-Sequenzierung seit etlichen Jahren erfolgreich angewandt, um biologische Prozesse zu untersuchen – jedoch ist deren Eignung für Infektionsstudien in starkem Maße limitiert. Die vorliegende Arbeit beschreibt die Etablierung eines neuartigen Ansatzes, „Duale RNA-Sequenzierung“ genannt, der Transkriptomstudien mit der Infektionsbiologie kompatibel macht. Mithilfe dieser Technologie wurde hier im Besonderen versucht, die Rolle nicht-proteinkodierender RNA-Moleküle für die Virulenz zu beleuchten, da die Charakterisierung dieser RNA-Klassen bisherigen Infektionsstudien weitgehend verwehrt blieb. Die Anwendbar-keit der Dualen RNA-Sequenzierung wurde innerhalb eines In-vitro-Infektionsmodells getestet, welches auf dem wichtigen, fakultativ intrazellulären Pathogen Salmonella enterica serovar Tyhimurium und verschiedenen humanen Zelllinien basiert. Die Duale RNA-Sequenzierung zeigte sich dabei in der Lage alle wesentlichen bakteriellen sowie humanen Transkriptklassen zu erfassen und erwies sich als reproduzierbar. Im Zuge dieser Experimente wurde ein Gen für eine zuvor kaum beschriebene kleine nicht-kodierende RNA (STnc440) als eines der am stärksten induzierten Gene intrazellulärer Salmonellen identifiziert. Interessanterweise hatten vorherige Studien gezeigt, dass die Inaktivierung dieses Gens zu einem Virulenzdefizit innerhalb unterschiedlicher Tiermodelle für Salmonellose führt. Die zugrunde liegenden molekularen Mechanismen blieben jedoch unbekannt. In der vorliegenden Arbeit wurden genetische, Transkriptom- sowie biochemische Analysen eingesetzt um das komplexe Regulationsnetzwerk dieser kleinen RNA erstmals näher zu beleuchten. Im Einzelnen konnte gezeigt werden, dass STnc440 die Expression mehrerer bakterieller mRNAs durch das Ausbilden zwischen-molekularer Basenpaarungen reguliert, was weitreichende Konsequenzen sowohl für die Virulenz des Pathogens als auch die Immunantwort des Wirts hat. Diese Ergebnisse veranschaulichen das Potential der Dualen RNA-Sequenzierung für das Auffinden und Charakterisieren neuer bakterieller Virulenzfaktoren während der Wirtsinfektion.

No description available.

Live attenuated Salmonella vectors provide a unique alternative in terms of antigen presentation by acting as a vector for heterologous antigens. The efficiency of any live bacterial vector rests with its ability to present sufficient foreign antigen to the human or animal immune system to initiate the desirable protective immune response. Salmonella vectors encoding heterologous protective antigens can elicit the relevant immune responses, be it humoral, mucosal or cell-mediated. STM-1 is a Salmonella mutant developed by RMIT, harbours a mutation in the aroA gene that renders it attenuated, and is a well characterised vaccine strain currently in use to protect livestock against Salmonella infection. In previous work in this laboratory, STM1 was shown to be capable of eliciting immune responses in mice to plasmid-borne antigens. In this study STM-1 was analysed for its ability to vector the model antigen chicken ovalbumin and test antigen C. jejuni major outer membrane protein using in vivo inducible promoters such as pagC and nirB from the plasmid location. The determination of the architecture around the lesion in STM-1 also allowed the development of constructs expressing heterologous antigen from the chromosome. The induction of immune responses, both humoral and cell mediated, was analysed. Another issue addressed in this study was effect of pre-existing immune responses in the animal host against the vector or related strains and the effects on generation of immune responses against the subsequently vectored antigen. Humoral and cellular immune responses to vectored ovalbumin and C. jejuni Momp antigens were observed following vaccination with STM-1, when antigens were expressed from either the plasmid or chromosomal location. Up-regulation of immune responses, both humoral and cell mediated, was observed against the vectored antigens in animals which were pre-exposed to either the bacterial vector or related strains. These results indicate that STM-1 has the potential to be used as a vector to deliver heterologous vaccine antigens from a single copy gene in the field. Lastly, the results from this study indicate that pre-existing immune responses against the bacterial vector or a related strain do in fact enhance both humoral and T cell responses against the heterologous antigen.

<p>An analysis of the recorded cases of Salmonella in Sweden indicates that there was no significant increase between 1997-2008, including or excluding outbreaks. The most common serotypes of Salmonella in Sweden were found to be S. Typhimurium and S. Enteritidis, with only the incidence of S. Typhimurium significantly increasing between 1997-2008. The most common phage types identified were S. Typhimurium 40, 104, NST and NT, with both S. Typhimurium NST and NT found to be significantly increasing. The season that has the greatest incidence of salmonellosis is the summer, from June to August. The number of Salmonella outbreaks occurring each year was found to be statistically increasing, this was however almost certainly due to a change in cluster detection techniques introduced in 2008, since a re-analysis excluding this year found no significant increase between 1997-2007. The most common serotypes that caused outbreaks during the study period were found to be S. Typhimurium, S Enteritidis, S. Saintpaul, S. Stanley and S. Hadar. There was no relationship found between the serotypes, with the number of cases not being proportional to the number of outbreaks. Despite these positive results, it should be of concern that evidence was found indicating that the sources of Salmonella infection appear to be changing, with an increase in the presence of ‘non-domestic’ serotypes.</p><p> </p>

Salmonella outbreaks associated with orange juices have been reported in the past. Though there have been studies on the internalization of Salmonella into oranges there is inadequate information on the ability of this pathogen to survive on orange surfaces, become internalized, and survive the low pH internal conditions. The objective of this work was to study the survival of Salmonella gaminara and Salmonella agona on oranges obtained from the field and retail outlets and investigate their attachment and internalization potential. These studies showed that oranges obtained from both the field and retail outlets harbored relatively high concentrations of aerobic heterotrophic bacterial populations. There were significant differences in the survival of Salmonella agona and Salmonella gaminara at 4??C, room temperature (25??C) and 37??C. Survival was highest at 37??C and lowest at 4??C for both Salmonella gaminara and Salmonella agona. Salmonella agona and Salmonella gaminara showed significant differences in recovery when the cells were treated with pH 4.0, 7.0 and 9.5 buffers. The internalization studies suggest that a negative temperature differential favors the internalization of Salmonella cells into the fruit. Significant differences in the internalization of Salmonella into field and market oranges were observed with more internalization in the field oranges as compared to the market oranges. These results suggest that to prevent Salmonella contamination of orange juices adequate pre-harvest protection against pathogen contamination and post-harvest cleaning and disinfection strategies need to be employed.

This study measured and compared different temperatures and dwell times of hot water treatment on the reduction of Escherichia coli O157:H7 and Salmonella Typhimurium on beef carcass surfaces. Two different types of beef surfaces, lean and fat, were inoculated with a fecal slurry containing E. coli O157:H7 and S. Typhimurium at ca. 7-log CFU/g, washed to remove gross fecal matter, and rinsed with hot water between 66 and 82 degrees C (150 to 180 degrees F water) for either 5, 10, or 15 s. There were no differences (P > 0.05) in the log reductions of S. Typhimurium and E. coli O157:H7 on the lean surfaces for all three temperature treatments (66, 74, and 82 degrees C). Although the 15 s treatment resulted in a numerically higher log reduction than the other treatments, each of the times resulted in at least a 1 log reduction of both S. Typhimurium and E. coli O157:H7 for lean surfaces. For the fat surfaces, all time treatments for the 82 degrees C and the 10 and 15 s treatments for the 74 degrees C resulted in the highest log reduction for S. Typhimurium. The 5 and 10 s dwell times for treatments at 66 degrees C and the 5 s dwell time at 74 degrees C resulted in the lowest log reduction of S. Typhimurium and E. coli O157:H7. For E. coli O157:H7 all temperature and time treatments resulted in at least a 1 log reduction for the fat surfaces of the outside round. Therefore, hot water treatment is a proven method for reducing both coliforms and pathogenic bacteria.