No description available.

No description available.

No description available.

Vibrio parahaemolyticus is a food-borne pathogen commonly associated with the consumption of raw or undercooked seafood resulting in primary infections of the human gastrointestinal tract. It is estimated to cause about 4500 illnesses each year in the United States. However, infection from this food-borne pathogen can be avoided if this organism is detected in the implicated food, prior to consumption. Current standard methods of detecting this organism are dependent on the culturability of the bacteria. Detection based on an organism’s culturability may be problematic as V. parahaemolyticus has been known to exist in a viable but nonculturable (VBNC) state. Bacteria in the VBNC state are characterized by low levels of metabolic activity and the inability to be cultured by standard laboratory practices. When bacteria enter the VBNC state, their gene expression profile may be different than the culturable counterpart. We were interested in comparing the expression of two virulence-associated genes between VBNC and culturable cells of V. parahaemolyticus. V. parahaemolyticus RIMD2210633 was incubated at 4°C in modified Morita mineral salt solution supplemented with 0.5% NaCl (MMS) or trypticase soy broth supplemented with 2% NaCl (TSBS), which represented nutrient poor and rich conditions, respectively. The number of VBNC and culturable cells were determined by standard plate count and fluorescence microscopy. The expression levels of virulence-associated genes tdh2 and escU, were measured relative to the housekeeping gene, pvsA, by qRT-PCR. Nutrient availability and temperatures exerted variable effects on the virulence gene expression. It is possible that VBNC V. parahaemolyticus cells may retain their pathogenicity potential.

This study was conducted to examine the rate of contamination and molecular characteristics of enteric bacteria isolated from a selection of food sources in Vietnam. One hundred and eighty raw food samples were tested and 60.8% of meat and 18.0% of shellfish samples were found to be contaminated with Salmonella spp. which belonged to variety of serogroups and serotypes. More than 90% of all food sources contained Escherichia coli and 32% of 50 shellfish samples were contaminated with Vibrio parahaemolyticus. PFGE was used to determine the degree of relatedness of Salmonella spp. There were 33 distinct PFGE patterns from 51 Salmonella spp. isolates tested, indicating that PFGE could be used as an alternative method for serotyping for use in epidemiology of Salmonella spp. Susceptibility of the isolates to 15 antimicrobial agents was investigated. Moderate to high frequencies of resistance to antibiotics were observed in Salmonella spp. and E. coli isolates and multi-resistance, defined as resistance to at least 4 antibiotics, was observed. All of the V. parahaemolyticus isolates were resistant to ampicillin/amoxicillin but not to other antibiotics. Betalactam TEM gene and tetracycline resistance tetA, tetB genes were widely distributed in both E. coli and Salmonella spp. isolates. Other resistance genes, including sulI, cmlA, aadA, aphA1, dhfrV, and aac(3)-IV were also present at high to moderate levels. Identification and characterisation of the mobile genetic elements, including identification of class 1 integrons and plasmids were carried out for multi-resistance isolates. The integrons harboured varying gene cassettes, including aadA1, aadA2, aadA5, aacA4, dhfrXII, drfA1 and dhfrA17, blaPSE1 and catB3. Thirty-five percent of Salmonella spp. isolates and 76% of E. coli isolates harboured plasmids of more than 95 kb. Transfer of resistance phenotypes between the isolates via conjugation and phage transduction was also demonstrated. Salmonella genomic island 1 (SGI1), a 43-kb genomic region contains a 13-kb antibiotic resistance gene cluster, has been identified in an isolate of S. Albany from chicken. The presence of Salmonella spp. virulence genes was investigated to examine the pathogenicity potential of the isolates. The invA gene was present in all Salmonella spp. isolates and the plasmid virulence gene spvC was detected in one S. Typhimurium isolate only, on a 95 kb virulence plasmid. Invasion assays performed in vitro demonstrated that all Salmonella isolates were capable of invading human intestine INT407 cells. In addition, the investigation for the presence of 58 selected virulence genes showed that all the tested isolates contained at least one virulence gene and there were 16 genes which are associated with different pathotypes detected. The data obtained in this study indicates that raw food in Vietnam is a potential reservoirs for many pathogenic organisms, and confirms the role of food animals as a reservoir of multidrug resistant E. coli and Salmonella spp.

Physicians frequently face challenges in predicting which bacterial subpopulations are likely to cause severe infections. A more accurate prediction of virulence would improve diagnostics and limit the extent of antibiotic resistance. Nowadays, bacterial pathogens can be typed with high accuracy with advanced genotyping technologies. However, effective translation of bacterial genotyping data into assessments of clinical risk remains largely unexplored. The discovery of unknown virulence genes is another key determinant of successful prediction of infectious disease outcomes. The trial-and-error method for virulence gene discovery is time-consuming and resource-intensive. Selecting candidate genes with higher precision can thus reduce the number of futile trials. Several in silico candidate gene prioritisation (CGP) methods have been proposed to aid the search for genes responsible for inherited diseases in human. It remains uninvestigated as to how the CGP concept can assist with virulence gene discovery in bacterial pathogens. The main contribution of this thesis is to demonstrate the value of translational bioinformatics methods to address challenges in virulence prediction and virulence gene discovery. This thesis studied an important perinatal bacterial pathogen, group B streptococcus (GBS), the leading cause of neonatal sepsis and meningitis in developed countries. While several antibiotic prophylactic programs have successfully reduced the number of early-onset neonatal diseases (infections that occur within 7 days of life), the prevalence of late-onset infections (infections that occur between 7??30 days of life) remained constant. In addition, the widespread use of intrapartum prophylactic antibiotics may introduce undue risk of penicillin allergy and may trigger the development of antibiotic-resistant microorganisms. To minimising such potential harm, a more targeted approach of antibiotic use is required. Distinguish virulent GBS strains from colonising counterparts thus lays the cornerstone of achieving the goal of tailored therapy. There are three aims of this thesis: 1. Prediction of virulence by analysis of bacterial genotype data: To identify markers that may be associated with GBS virulence, statistical analysis was performed on GBS genotype data consisting of 780 invasive and 132 colonising S. agalactiae isolates. From a panel of 18 molecular markers studied, only alp3 gene (which encodes a surface protein antigen commonly associated with serotype V) showed an increased association with invasive diseases (OR=2.93, p=0.0003, Fisher??s exact test). Molecular serotype II (OR=10.0, p=0.0007) was found to have a significant association with early-onset neonatal disease when compared with late-onset diseases. To investigate whether clinical outcomes can be predicted by the panel of genotype markers, logistic regression and machine learning algorithms were applied to distinguish invasive isolates from colonising isolates. Nevertheless, the predictive analysis only yielded weak predictive power (area under ROC curve, AUC: 0.56??0.71, stratified 10-fold cross-validation). It was concluded that a definitive predictive relationship between the molecular markers and clinical outcomes may be lacking, and more discriminative markers of GBS virulence are needed to be investigated. 2. Development of two computational CGP methods to assist with functional discovery of prokaryotic genes: Two in silico CGP methods were developed based on comparative genomics: statistical CGP exploits the differences in gene frequency against phenotypic groups, while inductive CGP applies supervised machine learning to identify genes with similar occurrence patterns across a range of bacterial genomes. Three rediscovery experiments were carried out to evaluate the CGP methods: a) Rediscovery of peptidoglycan genes was attempted with 417 published bacterial genome sequences. Both CGP methods achieved their best AUC >0.911 in Escherichia coli K-12 and >0.978 Streptococcus agalactiae 2603 (SA-2603) genomes, with an average improvement in precision of >3.2-fold and a maximum of >27-fold using statistical CGP. A median AUC of >0.95 could still be achieved with as few as 10 genome examples in each group in the rediscovery of the peptidoglycan metabolism genes. b) A maximum of 109-fold improvement in precision was achieved in the rediscovery of anaerobic fermentation genes. c) In the rediscovery experiment with genes of 31 metabolic pathways in SA-2603, 14 pathways achieved an AUC >0.9 and 28 pathways achieved AUC >0.8 with the best inductive CGP algorithms. The results from the rediscovery experiments demonstrated that the two CGP methods can assist with the study of functionally uncategorised genomic regions and the discovery of bacterial gene-function relationships. 3. Application of the CGP methods to discover GBS virulence genes: Both statistical and inductive CGP were applied to assist with the discovery of unknown GBS virulence factors. Among a list of hypothetical protein genes, several highly-ranked genes were plausibly involved in molecular mechanisms in GBS pathogenesis, including several genes encoding family 8 glycosyltransferase, family 1 and family 2 glycosyltransferase, multiple adhesins, streptococcal neuraminidase, staphylokinase, and other factors that may have roles in contributing to GBS virulence. Such genes may be candidates for further biological validation. In addition, the co-occurrence of these genes with currently known virulence factors suggested that the virulence mechanisms of GBS in causing perinatal diseases are multifactorial. The procedure demonstrated in this prioritisation task should assist with the discovery of virulence genes in other pathogenic bacteria.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

Avian pathogenic Escherichia. coli (APEC) and Campylobacter are pathogenic threats to poultry and human health, respectively. In this study, the prevalence of these pathogens in Mississippi broilers and their antimicrobial resistance (AMR) properties were investigated, and a multidrug-resistant APEC strain (APEC-O2-MS1170) was further explored by whole-genome sequencing (WGS). The efficacy of in ovo injection of Lactobacillus in reducing the APEC in broilers was evaluated. Results revealed a high prevalence of APEC and Campylobacter in broilers and broiler products. A lot of isolates were resistant to antibiotics of different sorts. Moreover, the in ovo administration of Lactobacillus did not reduce the incidence of APEC. The WGS of APEC-O2-MS1170 revealed its detailed AMR and virulence properties and alerted a potential zoonotic risk. In conclusion, the Lactobacillus did not reduce the incidence of APEC in broilers, and the prevalence and AMR of APEC and Campylobacter are still challenges faced by the poultry industry.

<p>Availability of genome sequences for numerous bacterial species comprising of different bacterial strains allows elucidation of species and strain specific adaptations that facilitate their survival in widely fluctuating micro-environments and enhance their pathogenic potential. Different bacterial species use different strategies in their pathogenesis and the pathogenic potential of a bacterial species is dependent on its genomic complement of virulence factors. A bacterial virulence factor, within the context of this study, is defined as any endogenous protein product encoded by a gene that aids in the adhesion, invasion, colonization, persistence and pathogenesis of a bacterium within a host. Anecdotal evidence suggests that bacterial virulence genes are undergoing diversifying evolution to counteract the rapid adaptability of its host&rsquo / s immune defences. Genome sequences of pathogenic bacterial species and strains provide unique opportunities to study the action of diversifying selection operating on different classes of bacterial genes.</p>

<p>Availability of genome sequences for numerous bacterial species comprising of different bacterial strains allows elucidation of species and strain specific adaptations that facilitate their survival in widely fluctuating micro-environments and enhance their pathogenic potential. Different bacterial species use different strategies in their pathogenesis and the pathogenic potential of a bacterial species is dependent on its genomic complement of virulence factors. A bacterial virulence factor, within the context of this study, is defined as any endogenous protein product encoded by a gene that aids in the adhesion, invasion, colonization, persistence and pathogenesis of a bacterium within a host. Anecdotal evidence suggests that bacterial virulence genes are undergoing diversifying evolution to counteract the rapid adaptability of its host&rsquo / s immune defences. Genome sequences of pathogenic bacterial species and strains provide unique opportunities to study the action of diversifying selection operating on different classes of bacterial genes.</p>