Neisseria meningitidis is a major cause of septicemia and meningitis worldwide. Traditional typing methods like pulsed-field gel electrophoresis (PFGE) for identifying outbreaks are subjective and time consuming. Multi-locus variable number tandem repeats analysis (MLVA) is an objective typing method amenable to automation that has been used to type other bacterial pathogens. This report describes the development of MLVA for outbreak detection of N. meningitidis. Tandem Repeats Finder software was used to identify variable number tandem repeats (VNTRs) from 3 sequenced N. meningitidis genomes. PCR amplification of identified VNTRs was performed on DNA from 7 serogroup representative isolates. PCR products were sequenced and repeats were manually counted. VNTR loci identified by this screen were evaluated on a collection of 46 outbreak and sporadic serogroup C isolates. Alleles at each locus were concatenated to define the MLVA type for each isolate. Minimum spanning tree (MST) analysis was performed to determine the genetic relationships among the isolates. The genetic distance was defined as the summed tandem repeat difference (STRD) between isolates MLVA types. Outbreak clusters were defined by a STRD less than or equal to 3. These data was compared to PFGE data to determine the utility of MLVA for outbreak detection. Twenty-one VNTR loci with variable copy numbers among the sequenced genomes were identified that met the established criteria of short repeat length and consensus sequence > 85%. Seven VNTR loci were reliably amplified among the 7 serogroups tested. These loci had repeat lengths between 4 and 20 nucleotides and exhibited between 10 and 26 alleles among 61 isolates belonging to 7 different serogroups. MST analysis with 7 loci differentiated serogroups, discriminated sporadic isolates and identified 7 out of 8 serogroup C outbreaks. In summary, MLVA with 5 VNTR loci distinguished N. meningitidis isolates from 7 different serogroups and sporadic isolates within each serogroup. In addition, MLVA identified 88% of PFGE-defined serogroup C outbreaks. Further investigation of these and other outbreak-associated isolates is necessary to define the optimal combination of VNTR loci and to evaluate MST analysis criteria in order to determine the utility of MLVA for N. meningitidis outbreak detection.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-04122006-174415 |
| Date | 07 June 2006 |
| Creators | Price, Alicia Anne |
| Contributors | Catherine McEllistrem, Saleem Khan, Lee H. Harrison, Jeremy Martinson |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-04122006-174415/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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