Inbreeding ratio and genetic relationships among strains of the Western clawed frog, Xenopus tropicalis

The Western clawed frog, Xenopus tropicalis, is a highly promising model amphibian, especially in developmental and physiological research, and as a tool for understanding disease. It was originally found in the West African rainforest belt, and was introduced to the research community in the 1990s. The major strains thus far known include the Nigerian and Ivory Coast strains. However, due to its short history as an experimental animal, the genetic relationship among the various strains has not yet been clarified, and establishment of inbred strains has not yet been achieved. Since 2003 the Institute for Amphibian Biology (IAB), Hiroshima University has maintained stocks of multiple X. tropicalis strains and conducted consecutive breeding as part of the National BioResource Project. In the present study we investigated the inbreeding ratio and genetic relationship of four inbred strains at IAB, as well as stocks from other institutions, using highly polymorphic microsatellite markers and mitochondrial haplotypes. Our results show successive reduction of heterozygosity in the genome of the IAB inbred strains. The Ivory Coast strains clearly differed from the Nigerian strains genetically, and three subgroups were identified within both the Nigerian and Ivory Coast strains. It is noteworthy that the Ivory Coast strains have an evolutionary divergent genetic background. Our results serve as a guide for the most effective use of X. tropicalis strains, and the long-term maintenance of multiple strains will contribute to further research efforts

Genetic diversity among Frankia strains nodulating members of the family Casuarinaceae in Australia revealed by PCR and restriction fragment length polymorphism analysis with crushed root nodules

DNA extracted directly from nodules was used to assess the genetic diversity of #Frankia# strains symbiotically associated with two species of genus #Casuarina# and two of the genus #Allocasuarina# naturally occuring in northeastern Australia. DNA from field-collected nodules or extracted from reference cultures of #Casuarina#-infective #Frankia# strains was used as the template in PCRs with primers targeting two DNA regions, one in the ribosomal operon and the other in the #nif# operon. PCR products were then analysed by using a set of restriction andonucleases. Five distinct genetic groups were recognized on the basis of these restriction patterns. These groups were consistently associated with the host species from which the nodules originated. All isolated reference strains had similar patterns and were assigned to group 1 along with six of eight unisolated #Frankia# strains from #Casuarina equisetifolia# in Australia. Group 2 consisted of two unisolated #Frankia# strains from #C. equisetifolia#, whereas groups 3 to 5 comprised all unisolated strains from #Casuarina cunninghamiana#, #Allocasuarina torulosa#, and #Allocasuarina littoralis#, respectively. These results demonstrate that, contrary to the results previous molecular studies of isolated strains, there is genetic diversity among #Frankia# strains that infect members of the family Casuarinaceae. The apparent high homogeneity of #Frankia# strains in these previous studies probably relates to the single host species from which the strains were obtained and the origin of these strains from areas outside the natural geographic range of members of the family Casuarinaceae, where genetic diversity could be lower than in Australia. (Résumé d'auteur

Variations in type III effector repertoires do not correlate with differences in pathological phenotypes and host range observed for Xanthomonas citri pv. citri pathotypes

Xanthomonas citri pv. citri (Xac) is a quarantine bacterium causing Asiatic citrus canker. Strains of Xac are classified as pathogenic variants i.e. pathotypes, according to their host range: strains of pathotype A infect a wide range of rutaceous species, whereas strains of pathotype A*/Aw infect a restricted host range consisting of Mexican lime (C. aurantifolia) and alemow (C. macrophylla). Based on a collection of 55 strains we investigated the role of type III effectors (T3E) in host specialization. By PCR we screened 56 Xanthomonas T3Es and showed that Xac possesses a repertoire of 28 effectors, 24 of which are shared by all strains, while 4 (xopAI, xopAD, xopAG and xopC1) are present only in some A*/ Aw strains. However, their distribution could not account for host specialization. XopAG is present in all Aw strains, but also in three A* strains genetically distant from Aw , and all xopAG-containing strains induced HR-like reactions on grapefruit and sweet orange. A strains are genetically less diverse, induce identical phenotypic responses, and share exactly the same T3Es. Conversely, A*/ Aw strains exhibited a wider genetic diversity in which clades correlated to geographical origin and T3Es repertoire but not to pathogenicity. A*/Aw strains showed a broad range of reactions on several Citrus, but genetically related strains did not share phenotypic responses. Our results showed that A*/Aw strains are more variable (genetically and pathogenetically) than initially expected and that this variability should not be ignored when trying to describe mechanisms involved in the pathogen evolution and host specialization. (Texte intégral

Acquisition of an Agrobacterium Ri Plasmid and Pathogenicity by Other -Proteobacteria in Cucumber and Tomato Crops Affected by Root Mat

Root mat of cucumbers and tomatoes has previously been shown to be caused by Agrobacterium radiobacter strains harboring a root-inducing Ri plasmid (pRi). Nine other pRi-harboring -Proteobacteria have subsequently been isolated from root mat-infected crops. Fatty acid profiling and partial 16S rRNA sequence analysis identified three of these strains as being in the genus Ochrobactrum, five as being in the genus Rhizobium, and one as being in the genus Sinorhizobium. An in vitro pathogenicity test involving inoculation of cucumber cotyledons was developed. All pRi-harboring -Proteobacteria induced typical root mat symptoms from the cotyledons. Average transformation rates for rhizogenic Ochrobactrum (46%) and Rhizobium (44%) strains were lower than those observed for rhizogenic A. radiobacter strains (64%). However, individual strains from these three genera all had transformation rates comparable to those observed from cotyledons inoculatedwith a rhizogenic Sinorhizobium strain (75%)

Multilocus sequence types of invasive Corynebacterium diphtheriae isolated in the Rio de Janeiro urban area, Brazil

Invasive infections caused by Corynebacterium diphtheriae in vaccinated and non-vaccinated individuals have been reported increasingly. In this study we used multilocus sequence typing (MLST) to study genetic relationships between six invasive strains of this bacterium isolated solely in the urban area of Rio de Janeiro, Brazil, during a 10-year period. Of note, all the strains rendered negative results in PCR reactions for the tox gene, and four strains presented an atypical sucrose-fermenting ability. Five strains represented new sequence types. MLST results did not support the hypothesis that invasive (sucrose-positive) strains of C. diphtheriae are part of a single clonal complex. Instead, one of the main findings of the study was that such strains can be normally found in clonal complexes with strains related to non-invasive disease. Comparative analyses with C. diphtheriae isolated in different countries provided further information on the geographical circulation of some sequence types

Identification and characterisation of enteroaggregative Escherichia coli subtypes associated with human disease

Enteroaggregative E. coli (EAEC) are a major cause of diarrhoea worldwide. Due to their heterogeneity and carriage in healthy individuals, identification of diagnostic virulence markers for pathogenic strains has been difficult. In this study, we have determined phenotypic and genotypic differences between EAEC strains of sequence types (STs) epidemiologically associated with asymptomatic carriage (ST31) and diarrhoeal disease (ST40). ST40 strains demonstrated significantly enhanced intestinal adherence, biofilm formation, and pro-inflammatory interleukin-8 secretion compared with ST31 isolates. This was independent of whether strains were derived from diarrhoea patients or healthy controls. Whole genome sequencing revealed differences in putative virulence genes encoding aggregative adherence fimbriae, E. coli common pilus, flagellin and EAEC heat-stable enterotoxin 1. Our results indicate that ST40 strains have a higher intrinsic potential of human pathogenesis due to a specific combination of virulence-related factors which promote host cell colonization and inflammation. These findings may contribute to the development of genotypic and/or phenotypic markers for EAEC strains of high virulence

Characterisation of dairy strains of Geobacillus stearothermophilus and a genomics insight into its growth and survival during dairy manufacture : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology at Massey University, Palmerston North, New Zealand

The thermophilic bacilli, such as G. stearothermophilus, are an important group of contaminants in the dairy industry. Although these bacilli are generally not pathogenic, their presence in dairy products is an indicator of poor hygiene and high numbers are unacceptable to customers. In addition, their growth may result in milk product defects caused by the production of acids or enzymes, potentially leding to off-flavours. These bacteria are able to grow in sections of dairy manufacturing plants where temperatures reach 40 – 65 °C. Furthermore, because they are spore formers, they are difficult to eliminate. In addition, they exhibit a fast growth rate and tend to readily form biofilms. Many strategies have been tested to prevent the formation of thermophilic bacilli biofilms in dairy manufacture, but with limited success. This is, in part, because little is known about the diversity of strains found in dairy manufacture, the structure of thermophilic bacilli biofilms and how these bacteria have adapted to grow in a dairy environment. In Chapters 2 and 3, phenotypic approaches were taken to understand the diversity of strains within a manufacturing plant. Specifically in Chapter 2, strains of the most dominant thermphilic bacilli, G. stearothermophilus, were isolated from the surface of various locations within the evaporator section and ten strains were evaluated for different phenotypic characteristics. Biochemical profiling, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and fatty profiling demonstrated that the population was diverse. In Chapter 3, it was shown that the same ten strains varied in their ability to form biofilms and produce spores. Three strains of G. stearothermophilus, A1, P3 and D1, were selected for further analysis. SEM demonstrated that there were differences in biofilm morphologies between the three strains, particularly D1 versus the other two strains, A1 and P3. In Chapters 4, 5 and 6 a comparative genomics approach was taken to determine how these bacteria are able to grow and survive within a dairy manufacturing environment, as well as how they differ from other strains of Geobacillus. In Chapter 4 draft genome sequences were generated for three strains of G.stearothermophilus. Identification of a putative lactose operon in the three dairy strains provided evidence of dairy adaptation. In Chapter 5 a phylogenomics approach was taken to resolve relationships within the Geobacillus genus and to identify differences within the G. stearothermophilus group itself. Finally in Chapter 6 comparison with the model organism B. subtilis, gave a genomics insight into the potential mechanisms of sporulation for Geobacillus spp

In vitro susceptibility of 120 strains of Neisseria gonorrhoeae isolated in Kyrghyzstan.

BACKGROUND: The World Health Organization has established a worldwide program for gonococcal antimicrobial surveillance, but so far no data on gonococcal susceptibility in Central Asia are available. GOAL: The need for biological data on the susceptibility of Neisseria gonorrhoeae in Kyrghyzstan, to enable adaptation of the national treatment protocol for gonococcal infections, led Médecins Sans Frontières and Epicentre to conduct a survey in collaboration with the Alfred Fournier Institute in Paris and the health authorities in Bishkek. STUDY DESIGN: In vitro susceptibility of N gonorrhoeae strains was determined with use of the reference agar-plate dilution technique. RESULTS: Results for 11 antibiotics tested on 120 strains of gonococci showed a low proportion (11.7%) of penicillinase-producing N gonorrhoeae and high proportions of intermediate or resistant strains to the majority of the antibiotics tested, including fluoroquinolones (>or=25% of strains resistant). All the strains were susceptible to spectinomycin, and only two strains had decreased susceptibility to cefixime. CONCLUSION: The therapeutic choices available in Kyrghyzstan appear to be limited to cephalosporins and spectinomycin

Bicarbonate Resensitization of Methicillin-Resistant Staphylococcus aureus to β-Lactam Antibiotics.

Endovascular infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a major health care concern, especially infective endocarditis (IE). Standard antimicrobial susceptibility testing (AST) defines most MRSA strains as "resistant" to β-lactams, often leading to the use of costly and/or toxic treatment regimens. In this investigation, five prototype MRSA strains, representing the range of genotypes in current clinical circulation, were studied. We identified two distinct MRSA phenotypes upon AST using standard media, with or without sodium bicarbonate (NaHCO3) supplementation: one highly susceptible to the antistaphylococcal β-lactams oxacillin and cefazolin (NaHCO3 responsive) and one resistant to such agents (NaHCO3 nonresponsive). These phenotypes accurately predicted clearance profiles of MRSA from target tissues in experimental MRSA IE treated with each β-lactam. Mechanistically, NaHCO3 reduced the expression of two key genes involved in the MRSA phenotype, mecA and sarA, leading to decreased production of penicillin-binding protein 2a (that mediates methicillin resistance), in NaHCO3-responsive (but not in NaHCO3-nonresponsive) strains. Moreover, both cefazolin and oxacillin synergistically killed NaHCO3-responsive strains in the presence of the host defense antimicrobial peptide (LL-37) in NaHCO3-supplemented media. These findings suggest that AST of MRSA strains in NaHCO3-containing media may potentially identify infections caused by NaHCO3-responsive strains that are appropriate for β-lactam therapy