Effects of a 10% xylitol solution (X) on adhesion of Escherichia coli O157:H7 and Salmonella serotype Typhimurium to meat surfaces were examined utilizing three approaches. In Experiment 1, rifampicin-resistant strains of E. coli O157:H7 and S. Typhimurium were dispersed in xylitol or a peptone solution (containing approximately 8.9 mean log per ml of each pathogen) and used to inoculate beef outside round meat surfaces. Samples were then rinsed with water or not rinsed in a 2X2 factorial arrangement. No interaction existed between inoculum type and post-inoculation treatments (P > 0.84). Incubation of pathogens in peptone or xylitol had minimal impact on pathogen adhesion (P > 0.76). Rinsing reduced counts by approximately 0.5 log CFU/cm2 (P < 0.01). Experiment 2 meat samples received a pretreatment of a water rinse, xylitol, or no rinse, followed by inoculation with pathogens dispersed in peptone solution (containing approximately 8.6 log mean log per ml of each pathogen). Samples received a post-inoculation treatment of a water rinse, xylitol rinse or no rinse in a 3X3 factorial arrangement. No interactions between pre- and post-inoculation factors were observed for surface pathogen load (P > 0.50). Post-inoculation rinsing reduced counts by approximately 0.5 log CFU/cm2 (P < 0.01) with no difference between water and xylitol (P > 0.64). Experiment 3 carcass surfaces were inoculated with pathogens at an initial level of 5.5 log CFU/cm2 and received a hot (35°C) water wash, 2.5% L-lactic acid spray, 10% xylitol spray, lactic acid + xylitol or hot water + xylitol. Pathogen counts were taken at 0 and 24 h post treatment. Lactic acid treatments reduced Salmonella by 3.3 log CFU/cm2 at 0 h (P < 0.01) and by 2.6 log CFU/cm2 after 24 h (P < 0.02). Hot water treatments reduced Salmonella by 1.5 log CFU/cm2 at 0 h (P < 0.07). Xylitol did not minimize pathogens (P > 0.62) nor did it increase effectiveness of other treatments. These data indicate that xylitol is ineffective at preventing E. coli O157:H7 and S. Typhimurium adhesion to meat surfaces.

Zugl.: Berlin, Freie Universiẗat, Diss., 2009.

No description available.

In recent decades salmonellosis has been on the rise as a food related illness worldwide. Causing over 24% of all non-typhoidal Salmonellosis cases, SE is the most frequently isolated serovar of Salmonella. Increased isolation of SE from eggs has paralleled an increase in the number of transovarian infections associated with laying hens in the poultry industry. This route of infection is a fairly new line of study when compared to the more traditional path where SE originates from fecal contamination through the shell. Salmonella Typhimurium (ST) is another concern for the egg industry. ST has caused 23.5% of all non-typhoidal salmonellosis cases. Understanding these two egg pathogens requires an in depth look at the mechanisms by which an egg may support infection and bacterial growth. Eggs were inoculated with both SE and ST onto the vitelline membrane and incubated for 24 hours. It was hoped that by gathering samples from the interior of the egg membrane, the albumen of the egg, and the membrane itself, some clarification as to when Salmonella is allowed to grow within the egg could be gathered. Albumen and membrane were found to be more hospitable environments to bacterial growth with increased storage times. In order to better understand the movement of bacteria into pre-ovulatory tissues, samples were gathered from mature laying hens. Follicular tissues were separated into divisions based on maturity, and bacteria were added to an in vitro cell culture broth containing the follicles. The point of this experiment was to determine if either species of Salmonella preferentially moved into follicles of different maturity when inoculated in vitro. A third experiment looked into the role of developmental stages of the vitelline membrane in exclusion of bacteria from the nutrient rich yolk. Tissues were gathered in the method described above. The follicular sack was removed from half of these samples and left intact for the other half. Another treatment group included was the yolks of eggs which had been laid by the same flock of birds. Results showed that follicles with intact follicular sacks were more susceptible to bacterial colonization than other treatment groups.

Depuis quelques années, il y a émergence de souches de Salmonella enterica sérovar Typhimurium multirésistantes causant une septicémie et la mort chez le porc. Ceci constitue un problème majeur pour l’industrie porcine et possiblement pour la santé publique. L’objectif de ce projet était de comparer et de caractériser une souche capable de causer une septicémie chez le porc et une souche commensale, en observant l’interaction avec des cellules épithéliales, des macrophages humains et d’identifier des gènes exprimés par les souches septicémiques et les souches commensales. Tout d’abord, l’infection de cellules épithéliales permet d’observer l’adhérence et l’invasion des bactéries, pour ainsi mettre en évidence la capacité des souches à coloniser le tractus gastro-intestinal. La souche commensale possède un pouvoir d’adhésion supérieur à la souche septicémique. Par la suite, l’infection de macrophages permet de caractériser le niveau de phagocytose et de survie. L’importance de la survie dans les macrophages pourrait permettre de faire un lien avec la septicémie. Toutefois, aucune différence n’est observable dans les conditions qui ont été testé. Ensuite, la technique SCOTS (Selective Capture of Transcribed Sequences) est utilisée pour capturer des gènes uniques à la souche septicémique et un autre SCOTS est fait pour capturer les gènes spécifiques à la souche commensale. Finalement, les gènes sont clonés, leur spécificité face aux souches est analysé par dot blot et ils sont identifiés par séquençage suivient d’une analyses bioinformatiques. Les gènes identifiés par SCOTS, lors des captures pour la souche septicémique et la souche commensale, se trouvent à être des gènes communs aux Salmonella. Toutefois, la différence de pathologie causée par les deux souches, n’est peut-être pas l’acquisition de nouveaux gènes, mais plutôt une différence d’expression entre les deux souches. / For many years, there has been an emergence of new multi-drug resistant Salmonella enterica serovar Typhimurium strains responsible for high mortality rates in regards to swine infections. This represents a major problem for the swine industry and also for public health. The objective of this project was to characterize an isolate capable of creating swine septicemic illness and another which is naturally present in this host’s intestinal tract. The study conducted here includes observations of bacteria-epithelial cell interactions, bacteria-macrophage interactions and identification of genes expressed during different epithelial cell infections. Bacterial adherence and invasion of cells were observed after epithelial cell infection. This type of assay underlines the ability of the isolates to colonize the intestinal tract. The commensal strain has a better adherence then the other. Macrophage infections allowed characterization of bacterial phagocytosis and intracellular survival. Survival within macrophages reflects the capacity of the bacterial isolates to infect and survive in the host. There’s no difference between the two strains with the condition we test. A technique termed SCOTS (Selective Capture of Transcribed Sequences) was used to capture unique transcripts from the isolate which caused illness during swine infection. SCOTS was also used to find unique genes expressed by the commensal isolate. The captured fragments were cloned, their strain specificity was analyzed by dot blot and the genes transcribed were identified by direct sequencing followed by bioinformatic analysis. SCOTS carried out with the septicemic and the commensal isolates was done. During the SCOTS conducted with the strain isolated from a sick swine or with the isolate from healthy swine, common Salmonella genes were found. Thus, even though these two isolates cause different pathologies, virulence of the strains does not rely on the acquisition of new genes, but perhaps on differential gene expression among both.

A model of growth and plasmid transfer between strains of Escherichia coli and Salmonella typhimurium was developed with reference to the literature. This was the organising principle for the collection of a complete set of in vitro life history parameters of one S. typhimurium and one E. coli strain. In the course of estimating these parameters two results of note were obtained. Fits of the Lotka-Volterra competition model were obtained for data on S. typhimuiurm growing in competition with E. coli. The first noteworthy discovery was the failure of this model to account for several characteristics of growth of these strains under competition. The growth rates of plasmid-bearing and plasmid-free strains were obtained. The second main result came from examination of the results of the growth rate data, which revealed that the cost to S. typhimuiurm 576 of bearing the resistance plasmid was low (4%). The model was also used to simulate the effect of antibiotic dose on the density of the donor, recipient and transconjugant populations over time. These simulations predicted that there would be a convex relationship between antibiotic dose and transconjugant density (i.e. that the density would first rise, then fall, with increasing dose). Following from this result, laboratory experiments and in vivo experiments in chickens were directed towards obtaining information on the relationship between these two variables. This convex relationship was not demonstrated within a single experiment, although some experimental environments produced an increase in transconjugant density with dose, and others, a decrease. Few transconjugants were formed in vivo. In order to investigate the low cost of resistance and low rate of in vivo transconjugant production, cost of resistance and plasmid transfer rate of this plasmid in several strain combinations of E. coli and S. typhimuiurm was evaluated.

Most bacterial foodborne pathogens are shed intermittently from their animal hosts and are able to grow and persist in the environment. Cattle and pigs constitute the major animal reservoirs for these pathogens. The overall objective of this dissertation research was to improve understanding of the role of intermittent shedding and environmental persistence in the transmission and maintenance of Escherichia coli O157:H7 and Salmonella Typhimurium in their animal host populations. This objective was addressed through five interdepended studies. The study in Chapter II, describes the transmission of E. coli O157:H7 in a dairy herd using mathematical modeling that includes indirect transmission from the contaminated environment. The model predicts that the elevated ambient temperature during summer, together with the availability of large amount of drinking water per cattle, are the major factors for increased pathogen load in water and high prevalence of E. coli O157:H7 in cattle populations. The second study, in Chapter III, determined the variation in water-to-cattle ratios among feedlot pens and evaluated the association with the pen level management and environmental factors. Water-to-cattle ratio was found to vary greatly between feedlots and pens with lower water-to-cattle ratios on average had cooler drinking water. The study in Chapter IV, used a compartmental mathematical model of infection transmission, to evaluate the effect of cleaning on Salmonella Typhimurium control in a grower-finisher pig herd. Cleaning alone was not found to be an effective measure of control unless combined with other measures to reduce the level of bacterial shedding. The study in Chapter V, developed the multi-state Markov chain model to describe the fecal shedding pattern of three E. coli O157:H7 strains in cattle. One strain was not detected to shed, while the other two strains had on average different durations of host colonization, albeit not at the statistically significant level. The study in Chapter VI, used an experimental infection transmission approach to estimate and compare transmission rates for three different strains of E. coli O157:H7 in steers. The results revealed that the transmission rate of E. coli O157:H7 increases significantly with increasing levels of environmental contamination. Collectively, the five studies have highlighted the role of these pathogen characteristics in their transmission. The improved understanding of these characteristics will allow for better design of control measure to ensure food safety.

Thesis (Ph. D.)--University of Hawaii at Manoa, 1995. / Includes bibliographical references (leaves 120-131). / Microfiche. / xiii, 131 leaves, bound ill. 29 cm

Salmonella enterica serovar Typhimurium is a common cause of salmonellosis among humans and animals worldwide. In Australia, Typhimurium is responsible for over half of the salmonellosis cases. The Anderson phage-typing scheme is the primary means of long-term surveillance of Typhimurium outbreak isolates, and has played an important role in epidemiology. However, there exist quite a number of strains of Typhimurium that cannot be defined by the phage-typing scheme. Furthermore, the knowledge of evolutionary relationships among isolates of different phage types is still very limited and the genetic basis of phage type variation remains largely unknown. To address these issues, this study focused on molecular typing and evolution of Typhimurium. Fluorescent amplified-fragment length polymorphism (AFLP) was applied to 46 Typhimurium isolates comprising nine phage types in Australia using the restriction enzymes MseI and EcoRI and MseI +1 / EcoRI +1 primer pair combinations. The selected phage types, DT9, DT135, DT64, DT44, DT126, DT12a, DT1, DT141 and DT108, have been dominant or frequent phage types in animal and human infections in Australia in recent years. AFLP in the present study showed a very good discrimination power with Simpson index of diversity of 0.98, 35 different AFLP patterns were observed in the 46 isolates studied. The tree based on AFLP patterns showed good correlation with phage type, grouped most Typhimurium isolates by phage type, and differentiated all nine phage types. Furthermore, 84 phage-type specific polymorphic AFLP fragments, for which presence or absence correlated with phage type (including 25 with one exception to phage-type specificity) were observed in the 46 strains studied. Eighteen phage-type specific AFLP fragments were cloned and sequenced. Sixteen are of known genes or have a homologue in the databases. It was found a predominance of phage and plasmid genes rather than mutational changes in the AFLP fragments studied. Of the 18 cloned and sequenced AFLP fragments, only four relate to mutational changes in the S. enterica chromosome, the other 14 comprise DNA of mobile elements: nine are phage related, three are plasmid related and two are gain of DNA from unknown origin. Twelve of the 18 sequenced phage-type specific AFLP markers are polymorphic because the DNA is present or absent as indicated by Southern hybridization. Two of these markers were successfully used in preliminary PCR-based typing of 30 DT9 and 29 DT135 isolates from worldwide collections. 27 of the 30 DT9 isolates and all DT135 isolates tested were correctly categorized. The results implied a good potential to use the sequence of these fragments as the basis for a multiplex PCR or a microarray based molecular �phage� typing method for Typhimurium. This thesis also studied the molecular evolutionary relationships among the same set of 46 Typhimurium isolates using mutational changes detected by AFLP, or analysis of intergenic regions and their flanking genes in genome sequences. The complete genome sequence of Typhimurium LT2 was analysed by computer modelled AFLP. The polymorphic AFLP fragments, which matched with the modelled LT2 AFLP fragments, were amplified and sequenced by LT2 genome based primers to determine the changes. Forty-nine intergenic regions with higher pairwise differences between LT2 and Typhi CT18 were amplified and sequenced using LT2 genome based primers for one isolate of each phage type. 51 polymorphic sites were detected consisting of 18 in AFLP fragments and 33 in intergenic regions or their flanking genes. PCR-RFLP (restriction fragment length polymorphism) and SNaPshot were used to further investigate the distribution of the single nucleotide polymorphisms (SNPs) detected in intergenic regions in all isolates studied. Of the 18 mutational changes detected in AFLP fragments, eight were indels (insertions / deletions) and ten single base substitutions. Of the eight indels, four were in genes, three in intergenic regions, and one covered adjacent intergenic and coding regions. The four indels in genes all caused frameshift mutations, including three single base indels and one 19 bp deletion. Of the ten substitutions, one was in an intergenic region and nine in genes comprising three synonymous and six non-synonymous substitutions. Of the 33 polymorphic sites detected from sequences of 23 intergenic regions and their flanking genes, one was IS200 insertion and 32 single nucleotide polymorphisms (SNPs), of which 30 were single base substitutions and two were single base indels. Nine of the 33 variations were found in the flanking genes, which were all single base substitutions comprising four synonymous, four non-synonymous substitutions and one non-sense mutation. More non-synonymous than synonymous substitutions were found for those in coding regions within Typhimurium, indicating that slightly deleterious intraspecies mutations can be fixed within clones, such as various lineages of Typhimurium. The 51 polymorphic sites, which were inferred from sequences of both mutation related AFLP fragments, and intergenic regions and their flanking genes, gave a single phylogenetic tree of the 46 Typhimurium isolates studied. All sequences involved were compared with the homologous sequences in the available S. enterica genome sequences for serovars Typhi, Paratyphi A, Gallinarum, Enteritidis and Pullorum and this enabled the determination of the direction of the mutational changes in the isolates studied and the root of the phylogenetic tree. There were only two events inferred to have occurred twice, the remaining 49 polymorphisms can be explained by a single event. The data indicated that Typhimurium has a very strong clonal structure with a very low level of recombination over the time for diversification of Typhimurium as majority of clonal variations are from point mutations rather than recombination. The phylogenetic tree based on mutational changes showed that most Typhimurium isolates of a given phage type are in the same evolutionary group, but that some phage types appear to have arisen more than once. Comparison of the phylogenetic tree with AFLP data gave examples of unrelated isolates of a given phage type having common AFLP fragments comprising plasmid or phage genes, supporting the view that phage type can be determined by presence of specific phages or plasmids. The mutation-based tree showed that six of the nine phage types studied appeared to have a single origin, at least for the isolates studied. It also found that DT1 and DT44 had two independent origins even for the limited set of strains used. The distribution of DT12a isolates into two groups could be explained that the group of three DT12a isolates were derived from the other group of four DT12a isolates, where the root of the tree might be. The data also confirmed that DT64 arose from DT9. The phylogenetic tree that was generated based on essentially mutational changes provides clear relationships of the closely related Typhimurium isolates with high level of consistency and reasonable confidence. This study provided one of the few analyses of relationships of isolates within a clone. Matching actual AFLP with computer modeled AFLP and sequencing intergenic regions provide very good new strategies to identify mutational polymorphisms and to study the molecular evolutionary relationships in the closely related isolates.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.