The resistance of N. Molecular approaches are able to detect these alterations and to overcome technical problems of minimal inhibitory concentration MIC determination as well as culture failure.
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Indeed, 14 European Reference Laboratories using identical phenotypic methods, media and strains reported differences in MIC determinations. Beta lactams such as penicillin G are still the first line antibiotics in the treatment of invasive meningococcal infections. Alterations in the penA gene, encoding the penicillin binding protein 2 PBP2 , are directly linked to meningococcal reduced susceptibility to penicillin G Spratt, ; Antignac et al. The detection of the positions that are always modified in Pen I strains is a powerful tool to identify Pen I isolates and can be detected by molecular typing of the penA gene.
Sequencing of this part of penA seems to be the best method to predict Pen I phenotype Taha et al. To date, penA alleles have been identified. Alterations in the gyrA and parC genes of N. Transformation experiments have made it possible to ascertain that this mutation was directly responsible for the Rif R phenotype Taha et al. PCR amplification and sequencing can detect mutations in rpoB and can be used as a nonculture method to predict meningococcal resistance to rifampicin Stefanelli et al. Accurate nonculture strain characterization is essential for all levels of epidemiological surveillance to monitor any change in the populations of invasive meningococci following major intervention strategies such as vaccination.
Furthermore, for countries where large numbers of cases are nonculture infections, the ability to characterize meningococci directly from clinical material is essential for national surveillance.
This protocol has been fully evaluated on specimens from culture-proven cases and is based on nested PCR with a clinical sensitivity similar to that of real time PCR. The protocol is described in full in Birtles , including a full listing of all primer sets for both rounds of amplification and details of all amplification conditions. MLST is recommended for molecular typing of meningococcal isolates in reference laboratories, and the ability to perform nonculture MLST means that a single typing system is available for both culture and nonculture typing for meningococci. Serosubtyping is based on the antigenic specificity of the class 1 outer membrane protein, PorA serosubtype.
Genosubtyping N. Genosubtyping of meningococci is important for the development of vaccines specifically for serogroup B meningococci, and overcomes the problem of the decreasing typeability of meningococcal isolates by serosubtyping, whilst providing a high index of discrimination. Details of both first and second round primers and the associated amplification conditions are listed in detail in Birtles Furthermore, because of the improved typeability and discrimination achieved with porA sequence typing, the EMGM decided during its biannual assembly in Dublin that porA sequence typing should be implemented in all European reference laboratories as soon as possible.
This is an important step towards providing the same format for subtyping both culture- and nonculture-confirmed meningococcal infections. It should also allow integration of the European Invasive Bacterial Infections Surveillance network EU-IBIS database for pan-European surveillance, which currently receives both phenotypic and genotypic datasets from different countries.
PorA antigen sequence typing will not in every case or for every clonal lineage generate sufficient discriminatory power to support more advanced data mining such as computer-assisted cluster analysis Elias et al. The fetA frpB gene encoding the immunogenic iron-repressed siderophore receptor contains a plethora of polymorphic sites Thompson et al. Sequence typing of this variable region increases the number of finetypes achieved by serogrouping and PorA sequence typing alone Urwin et al.
The overall typeability is high, as only 0. At the German reference laboratory, FetA typing has been included in the culture and nonculture finetyping programme Vogel et al. For nonculture detection, DNA-extraction is accomplished using commercially available kits. Sensitivity of the amplification is controlled by dilutions of a cloned PCR product.
The test is approved if 10— copies of the plasmid in aqueous solution are detected. The sensitivity of the test is comparable to that of genogrouping and porA -typing. In summary, a complement to nonculture finetyping by serogrouping and PorA antigen typing is highly desirable; FetA antigen typing currently is the best candidate to be agreed upon in the near future by the European reference laboratories.
The interlaboratory collaboration between the European Meningococcal Reference laboratories as part of the EMGM has made a major contribution to the development and standardization of new methods for the improved management of meningococcal infection through improved case ascertainment and epidemiological characterization. Most recently, this has been given greater impetus through the EU-MenNet programme.
University College Cork
The development of methods for the full molecular characterization of meningococci both for culture- and nonculture-confirmed cases has provided the necessary tools for enhanced surveillance to manage the successful implementation of major disease prevention strategies across Europe. Molecular detection of genes associated with meningococcal resistance to antibiotics is now possible; this may overcome technical difficulties in interpreting classical antibiograms and can be applied directly on clinical samples for nonculture-confirmed cases.
In the next 2 years the EMGM will define polymorphic sites and establish culture-independent methods for identification, ensuring a comprehensive surveillance of emerging resistance also in patients who remain unconfirmed by culture. A database is expected to be available in with sequences of penA from a large collection of recent meningococcal isolates from several European countries as well as other countries. Moreover, this database should allow an objective determination of the breakpoint for penicillin G to define Pen I isolates.
There will be a move towards the widespread use of standardized genogrouping protocols and extraction methods for DNA isolation from clinical specimens either by individual reference laboratories or through collaboration and referral between centers over the next few years. Such distributions strains and clinical samples may be made annually through the EU-IBIS network to perform different molecular approaches that should be included in this regular EQA in addition to the usual conventional bacteriological methods:.
The laboratory and surveillance network now has the tools to influence the management and control of meningococcal infection to the benefit of the European community and beyond. Oxford University Press is a department of the University of Oxford. The oxidation of o -cresol at slower rate was also demonstrated.
The metabolite from o -cresol gave different UV absorption spectrum from that formed from 3-methylcatechol. From the cells of strains which belong to the genera Hansenula, Pichia , Citeromyces, Pachysolen , and Wingea , coenzyme Q Co-Q was extracted and partially purified. The type of Co-Q was determined mainly by paper chromatography. Others with the latter unique quinone system were Cit. In contrast, the heterogeneous nature of the genus Pichia was reconfirmed, because of a complex distribution of three different kinds of Co-Q Q 7 , Q 8 , and Q 9.
Most species of this genus with hat- or Saturn-shaped ascospores such as P. Pichia pastoris was the only species with Co-Q 8 in this genus. The Co-Q 9 system was found in P. The results concerning the Co-Q system are discussed in connection with other criteria such as serological characteristics, PMR spectra of cell-wall mannans, DNA base composition, etc. Most of the receptor activity was solubilized, by this procedure, and separated from spheroplast membrane fraction.
This suggested the location of receptor on the cell wall. Pyocin particles were adsorbed on the receptor site with the contracted sheath, at the tip of core. Pyocin R inactivation by its adsorption to the receptor was highly dependent on temperature, and the presence of 0.
Kinetic equations dealing with rates of substrate consumption, cell growth, respiration, product formation and heat evolution have been derived for Saccharonzyces cerevisiae growing on ethanol as the sole carbonaceous material. It is very important that inventors of antimicrobial wound dressings ensure efficacy against both planktonic and sessile microorganisms, within the in vitro and in vivo environments. Login to your account Username.
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