Binary typing of staphylococcus aureus

This thesis describes the development. application and validation of straindifferentiating DNA probes for the characterization of Staphylococcus aureus strains in a system. that yields a binary output. By comparing the differential hybridization of these DNA probes to staphylococcal genomes. further insight in the genomic flexibility. the evolutionary clock-speed. the route of transmission. or the possible role of pathogenicity of a given S. aureus strain can be obtained. The generation. and development of the strain-discriminating DNA probes. constituting the binary typing (BT) system. will be introduced in chapter II. Chapters III. IV and V provide examples of application of the BT system. The genetic diversity of methicillinresistant S. aureus (MRSA) strains as measured with the BT system is described in chapter III. The nationwide spread of a MRSA clone (chapter IV). and the epidemiology of bovine S. aureus strains (chapter V) was monitored with the BT system. and the results were compared with other genotyping approaches. The validation of the binary probes as stable epidemiological markers. described in chapter VI. was determined according to generally accepted evaluation parameters. The final technical protocol of the BT system is outlined in chapter VII. The feasibility and technical reproducibility of current BT protocol as measured in a multicenter study is presented in chapter VIII. Finally, chapter IX integrates the data presented in the previous chapters and provide future perspectives in the typing of S. aureus

Rapid detection of methicillin resistance in Staphylococcus aureus isolates by the MRSA-screen latex agglutination test

The slide agglutination test MRSA-Screen (Denka Seiken Co., Niigata, Japan) was compared with the mecA PCR ("gold standard") for the detection of methicillin resistance in Staphylococcus aureus. The MRSA-Screen test detected the penicillin-binding protein 2a (PBP2a) antigen in 87 of 90 genetically diverse methicillin-resistant S. aureus (MRSA) stock culture strains, leading to a sensitivity of 97%. The three discrepant MRSA strains displayed positive results only after induction of the mecA gene by exposure to methicillin. Both mecA PCR and MRSA-Screen displayed negative results among the methicillin-susceptible S. aureus strains (n = 106), as well as for Micrococcus spp. (n = 10), members of the family Enterobacteriaceae (n = 10), Streptococcus pneumoniae (n = 10), and Enterococcus spp. (n = 10) (specificity = 100%). Producing the same PBP2a antigen, all 10 methicillin-resistant Staphylococcus epidermidis strains score positived in both the latex test and the mecA PCR. Consequently, the MRSA-Screen test should be applied only after identification of the MRSA strain to the species level to rule out coagulase-negative staphylococci. In conclusion, due to excellent specificity and sensitivity the MRSA-Screen latex test has the potential to be successfully used for routine applications in the microbiology laboratory

Variable number of tandem repeats in clinical strains of Haemophilus influenzae

An algorithm capable of identifying short repeat motifs was developed and used to screen the whole genome sequence available for Haemophilus influenzae, since some of these repeats have been shown to affect bacterial virulence. Various di- to hexanucleotide repeats were identified, confirming and extending previous findings on the existence of variable-number-of-tandem-repeat loci (VNTRs). Repeats with units of 7 or 8 nucleotides were not encountered. For all of the 3- to 6-nucleotide repeats in the H. influenzae chromosome, PCR tests capable of detecting allelic polymorphisms were designed. Fourteen of 18 of the potential VNTRs were indeed highly polymorphic when different strains were screened. Two of the potential VNTRs appeared to be short and homogeneous in length; another one may be specific for the H. influenzae Rd strain only. One of the primer sets generated fingerprint-type DNA banding patterns. The various repeat types differed with respect to intrinsic stability as well. It was noted for separate colonies derived from a single clinical specimen or strains passaged for several weeks on chocolate agar plates that the lengths of the VNTRs did not change. When several strains from different patients infected during an outbreak of lung disease were analyzed, increased but limited variation was encountered in al

Genome sequence of Madurella mycetomatis mm55, isolated from a human mycetoma case in Sudan

We present the first genome sequence for a strain of the main mycetoma causative agent, Madurella mycetomatis. This 36.7-Mb genome sequence will offer new insights into the pathogenesis of mycetoma, and it will contribute to the development of better therapies for this neglected tropical disease

Fluorescently-labelled CPD and 6-4PP photolyases: new tools for live-cell DNA damage quantification and laser-assisted repair

UV light induces cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts (6-4PPs), which can result in carcinogenesis and aging, if not properly repaired by nucleotide excision repair (NER). Assays to determine DNA damage load and repair rates are invaluable tools for fundamental and clinical NER research. However, most current assays to quantify DNA damage and repair cannot be performed in real time. To overcome this limitation, we made use of the damage recognition characteristics of CPD and 6-4PP photolyases (PLs). Fluorescently-tagged PLs efficiently recognize UVinduced DNA damage without blocking NER activity, and therefore can be used as sensitive live-cell damage sensors. Importantly, FRAP-based assays showed that PLs bind to damaged DNA in a highly sensitive and dose-dependent manner, and can be used to quantify DNA damage load and to determine repair kinetics in real time. Additionally, PLs can instantly reverse DNA damage by 405 nm laserassisted photo-reactivation during live-cell imaging, opening new possibilities to study lesion-specific NER dynamics and cellular responses to damage removal. Our results show that fluorescently-tagged PLs can be used as a versatile tool to sense, quantify and repair DNA damage, and to study NER kinetics and UV-induced DNA damage response in living cells

Assessment of resolution and intercenter reproducibility of results of genotyping Staphylococcus aureus by pulsed-field gel electrophoresis of SmaI macrorestriction fragments: a multicenter study

Twenty well-characterized isolates of methicillin-resistant Staphylococcus aureus were used to study the optimal resolution and interlaboratory reproducibility of pulsed-field gel electrophoresis (PFGE) of DNA macrorestriction fragments. Five identical isolates (one PFGE type), 5 isolates that produced related PFGE subtypes, and 10 isolates with unique PFGE patterns were analyzed blindly in 12 different laboratories by in-house protocols. In several laboratories a standardized PFGE protocol with a commercial kit was applied successfully as well. Eight of the centers correctly identified the genetic homogeneity of the identical isolates by both the in-house and standard protocols. Four of 12 laboratories failed to produce interpretable data by the standardized protocol, due to technical problems (primarily plug preparation). With the five rel

Selective extraction of naturally occurring radioactive Ra2+

Organic extractants play a significant role in the selective removal of radioactive cations from waste streams. Although, literature on the selective removal of man-made radioactive material such as Americium (Am) is widespread, the selective removal of naturally occurring radioactive material such as Ra2+ is only mentioned sporadically. This tutorial review deals with the selective extraction of the highly radiotoxic Ra2+. Special attention is paid to different types of organic extractants used

Thiacalix(4)arene derivatives as radium ionophores: a study on the requirements for Ra2+ extraction

The synthesis and NOE-based structural characterization is described of thiacalix[4]arene tricarboxylic acid ( 7), thiacalix[4]crown-5 and -6 monocarboxylic acids ( 2 and 5), and the bis(N-methylsulfonyl)thiacalix[4]crowns-5 and -6 ( 4a,b). The 226Ra2+ selectivity coefficients, log(KRaex/KMex), of the new thiacalix[4]arene derivatives are compared directly with those of thiacalix[4]crown-5 and -6 ( 1a,b), thiacalix[4]crown-5 and -6 dicarboxylic acids ( 3a,b), and thiacalix[4]arene di- and tetracarboxylic acids ( 6 and 8). Thiacalix[4]arene dicarboxylic acid ( 6) already exhibits a high 226Ra2+ selectivity, but this is significantly improved in the case of 3b, having an additional crown-(6-)ether bridge. The covalent combination of a crown ether and carboxylic acid substituents as in the thiacalix[4]arenes 2, 3a,b, 4a,b, and 5 gives a better 226Ra2+ selectivity in the presence of Sr2+ or Ba2+ than mixtures of dibenzo-21-crown-7 and thiacalix[4]arene dicarboxylic acid ( 6) or of pentadecanoic acid and thiacalix[4]crown-6 ( 1b)

Cation control on the synthesis of p-t-butylthiacalix[4]-(bis)crown ethers

Bridging of p-t-butylthiacalix[4]arene with ethylene glycol ditosylates gave diametrically bridged thiacalix[4]monocrowns-4 and -5, 1,2-alternate thiacalix[4]biscrowns-4 and -5 and 1,3-alternate thiacalix[4]biscrown-5, dependent on the metal carbonate used. They show excellent extraction ability towards Ag+ cations.\ud The synthesis of novel diametrically and proximally substituted p-t-butylthiacalix[4](bis)crown ethers and their extraction behavior towards monovalent metal ions are presented

Synthesis and Conformational Evaluation of p-tert-Butylthiacalix[4]arene-crowns

Bridging of p-tert-butylthiacalix[4]arene afforded 1,3-dihydroxythiacalix[4]arene-monocrown-5 (3b), 1,2-alternate thiacalix[4]arene-biscrown-4 and -5 (4a,b), and 1,3-alternate thiacalix[4]arene-biscrown-5 and -6 (5a,b), depending on the metal carbonates and oligoethylene glycol ditosylates used. Starting from 1,3-dialkylated thiacalix[4]arenes, the corresponding bridging reaction gave 1,3-alternate, partial-cone, and cone conformers 10-19, depending on the substituents present. Temperature-dependent studies revealed that the conformationally flexible 1,3-dimethoxythiacalix[4]arene-crowns 10a-c exclusively occupy the 1,3-alternate conformation. Demethylation exclusively gave the cone 1,3-dihydroxythiacalix[4]arene-crowns (3a,c), which could not be obtained by direct bridging of thiacalix[4]arene. The different structures were assigned on the basis of several X-ray crystal structures and extensive 2-D 1H NMR studies