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ABSTRACT: If the acquisition of virulence genes (VGs) for pathogenicity were not solely acquired through horizontal gene transfers of pathogenicity islands, transposons, and phages, then clonal clusters of enterotoxigenic Escherichia coli (ETEC) would contain few or even none of the VGs found in strains responsible for extraintestinal infections. To evaluate this possibility, 47 postweaning diarrhea (PWD) ETEC strains from different geographical origins and 158 commensal E. coli isolates from the gastrointestinal tracts of eight group-housed healthy pigs were screened for 36 extraintestinal and 18 enteric VGs using multiplex PCR assays. Of 36 extraintestinal VGs, only 8 were detected (fimH, traT, fyuA, hlyA, kpsMtII, k5, iha, and ompT) in the ETEC collection. Among these, hlyA (alpha-hemolysin) and iha (nonhemagglutinating adhesin) occurred significantly more frequently among the ETEC isolates than in the commensal isolates. Clustering analysis based on the VG profiles separated commensal and ETEC isolates and even differentiated serogroup O141 from O149. On the other hand, pulsed-field gel electrophoresis (PFGE) successfully clustered ETEC isolates according to both serotype and geographical origin. In contrast, the commensal isolates were heterogeneous with respect to both serotype and DNA fingerprint. This study has validated the use of VG profiling to examine pathogenic relationships between porcine ETEC isolates. The clonal relationships of these isolates can be further clarified by PFGE fingerprinting. The presence of extraintestinal VGs in porcine ETEC confirmed the hypothesis that individual virulence gene acquisitions can occur concurrently against a background of horizontal gene transfers of pathogenicity islands. Over time, this could enable specific clonotypes to respond to host selection pressure and to evolve into new strains with increased virulence.
Applied and Environmental Microbiology 02/2007; 73(1):83-91. · 3.83 Impact Factor
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ABSTRACT: A combination of uni- and multiplex PCR assays targeting 58 virulence genes (VGs) associated with Escherichia coli strains causing intestinal and extraintestinal disease in humans and other mammals was used to analyze the VG repertoire of 23 commensal E. coli isolates from healthy pigs and 52 clinical isolates associated with porcine neonatal diarrhea (ND) and postweaning diarrhea (PWD). The relationship between the presence and absence of VGs was interrogated using three statistical methods. According to the generalized linear model, 17 of 58 VGs were found to be significant (P < 0.05) in distinguishing between commensal and clinical isolates. Nine of the 17 genes represented by iha, hlyA, aidA, east1, aah, fimH, iroN(E. coli), traT, and saa have not been previously identified as important VGs in clinical porcine isolates in Australia. The remaining eight VGs code for fimbriae (F4, F5, F18, and F41) and toxins (STa, STb, LT, and Stx2), normally associated with porcine enterotoxigenic E. coli. Agglomerative hierarchical algorithm analysis grouped E. coli strains into subclusters based primarily on their serogroup. Multivariate analyses of clonal relationships based on the 17 VGs were collapsed into two-dimensional space by principal coordinate analysis. PWD clones were distributed in two quadrants, separated from ND and commensal clones, which tended to cluster within one quadrant. Clonal subclusters within quadrants were highly correlated with serogroups. These methods of analysis provide different perspectives in our attempts to understand how commensal and clinical porcine enterotoxigenic E. coli strains have evolved and are engaged in the dynamic process of losing or acquiring VGs within the pig population.
Applied and Environmental Microbiology 08/2006; 72(7):4782-95. · 3.83 Impact Factor
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ABSTRACT: A group-specific primer pair was designed to amplify the 16S rRNA gene of representative reference strains from environmentally sourced, mesophilic aerobic spore-forming Bacillus taxa. The PCR generated a 1114 bp amplicon but did not do so with DNA extracted from 16 other Eubacterial species. When amplicons were digested with restriction enzymes AluI or TaqI, different profiles containing between 2 and 5 fragments ranging in size from 76 to 804 base pairs were seen with different Bacillus species. This procedure, known otherwise as amplified ribosomal DNA restriction analysis or ARDRA, produced unique and distinguishable patterns to differentiate between 15 ATCC reference strains (10 Bacillus, 3 Paenibacillus and 2 Brevibacillus member species) as well as 3 misidentified Bacillus probiotic strains in a commercial collection. Our simplified PCR-ARDRA protocol provides a facile method for the identification of most environmentally important species of Bacillus.
Journal of Microbiological Methods 02/2006; 64(1):107-19. · 2.09 Impact Factor
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ABSTRACT: Diversity studies of enteric Escherichia coli have relied almost entirely on faecal isolations on the assumption that they are representative of flora found throughout the gastrointestinal tract. The authors have addressed this belief by analysing isolates obtained from the duodenum, ileum, colon and faeces of pigs. E. coli isolates were obtained from eight pigs and characterized using multi-locus enzyme electrophoresis and PCR-based screening for a range of factors thought to be associated with intestinal and extra-intestinal disease. There are four main genetic groups of commensal E. coli (A, B1, B2, D). Group A strains represented 76 % of the isolates from the duodenum, ileum and colon compared to 58 % of the strains isolated from faeces. A nested molecular analysis of variance based on the allozyme and virulence factor screening results showed that differences among individual pigs accounted for 6 % of the observed genetic diversity, whilst 27 % of the genetic variation could be explained by clonal composition differences among gut regions. Finally, the absence of virulence genes in these commensals indicates that they may be suitable as a probiotic consortium, particularly if they also display increased adherence to enterocytes and antagonistic activity against pathogenic strains of E. coli.
Microbiology 07/2004; 150(Pt 6):1735-40. · 3.06 Impact Factor
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Xi-Yang Wu
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ABSTRACT: The mechanism of action of probiotics is based on competitive exclusion and immune modulation. However, the literature is scant on supporting data because of the failure to adopt a systems approach to probiotic functionality. This has been partially addressed in this thesis by taking into consideration the tripartite interaction between bacteria and bacteria in the enteric community; between bacteria and the host animal and finally, between the host immune response (innate or acquired) on the plethora of microbes that inhabit the gastrointestinal tract. A trial involving newly inducted cattle in a feedlot, formed the basis of initial attempts to assess the benefits of a commercial probiotic formulation - Protexin on intestinal health by enumeration of a select subset of cultivable bacteria species and by assessment of immune modulation. The results failed to demonstrate a significant change in the population dynamics of cultured faecal microbes but did show that Protexin stimulated immune responsiveness in T cells. Carcass analysis demonstrated a significant reduction in marbling or intramuscular fat deposition. In the course of examining the faecal microflora from feedlot cattle, the presence of high levels of Bacillus spores suggested that one possible reason for the lack of a growth benefit may be attributed to a high endogenous level of bacilli. Since there were no reliable methodologies for identifying Bacillus species, an alternative procedure was developed involving amplified ribosomal DNA restriction analysis (ARDRA). With this protocol, we were able to show that cattle faeces contained large numbers of Bacillus spores representing different mesophilic species, where B. subtilis, B. licheniformis and B. clausii dominated. The presence of a stable population of coliforms in cattle faeces that was not altered by probiotic feeding highlighted the importance of developing better techniques to characterise diversity in E. coli, a potential food-borne pathogen of economic significance to the cattle industry. The use of virulence genes to genotype coliforms provided a method for differentiating between pathogenic, clinical and commensal isolates of E. coli. Altogether, a combination of uni- and multiplex PCR assays was developed to screen for 50 virulence genes (VGs) from 8 pathotypes of E. coli. There was a significant association between phylogroupings and VG ownership. This result showed clearly that the lack of or possession of VGs in member isolates of each phylogenetic group can be used to assess diversity and potential pathogenesis of E. coli. To understand better the importance of pathogenic enteric coliforms, an alternative animal model involving pigs with post-weaning diarrhoea was used to investigate the relationship between pathogenicity and commensalism by VG profiling. Porcine enterotoxigenic E. coli (ETEC) were found to carry VGs identified in E. coli that cause extraintestinal infection. Furthermore, by using the appropriate methods of statistical analysis, VG profiling had the capacity to predict the pathogenic and commensal status of individual clones. By developing the capacity to rapidly characterise and genotype virulence and commensalism in E. coli, it is now feasible to examine how probiotic feeding can modulate the population dynamics of different community members in pigs with enteric disease, as well as changes in the coliform populations. Finally, another arm of the tripartite interaction involving bacteria and host interaction was modelled in vitro by examining the primary signalling events between bacteria and intestinal epithelial cells. These investigations focused on the judicious selection of T84 as the reporter intestinal epithelial cell line because of low level expression of inflammatory transcripts from 6 other epithelial cell lines. Using a panel of coliforms genotyped for virulence or lack of virulence, the signalling events that followed on from the primary interaction between bacterium and cell, showed there was a lack of correlation between VGs and gene activation. Nonetheless, all the coliform strains tested varied in their capacity to signal transduce T84, confirming that this differential bioactivity can be exploited in the ranking of candidate probiotic strains. The differential responses seen with different E. coli strains and the lower and more consistent activation patterns recorded by LABs for both cytokine and chemokine gene activation, demonstrate that a semi-quantitative ranking of microbial bioactivity can be obtained. Such an approach if adopted in conjunction with an even wider panel of genes in a standardised in vitro environment can provide invaluable information on the selection of appropriate strains to be further tested in vivo.
University of Wollongong Thesis Collection.
