Kimberlyn Nelson

STREPTOCOCCUS PYOGENES CAUSING TOXIC-SHOCK-LIKE SYNDROME AND OTHER INVASIVE DISEASES : CLONAL DIVERSITY AND PYROGENIC EXOTOXIN EXPRESSION

Genetic diversity and relationships among 108 isolates of the bacterium Streptococcus pyogenes recently recovered from patients in the United States with toxic-shock-like syndrome or other invasive diseases were estimated by multilocus enzyme electrophoresis. Thirty-three electrophoretic types (ETs), representing distinctive multilocus clonal genotypes, were identified, but nearly half the disease episodes, including more than two-thirds of the cases of toxic-shock-like syndrome, were caused by strains of two related clones (ET 1 and ET 2). These two clones were also represented by recent pathogenic European isolates. A previous report of a relatively high frequency of expression of exotoxin A among isolates recovered from toxic-shock-like syndrome patients in the United States was confirmed; and the demonstration of this association both within clones and among distantly related clones supports the hypothesis that exotoxin A is a causal factor in pathogenesis of this disease. Near identity of the nucleotide sequences of the exotoxin A structural gene of six isolates of five ETs in diverse phylogenetic lineages was interpreted as evidence that the gene has been horizontally distributed among clones, presumably by bacteriophage-mediated transfer.

Discrimination Amongst Leishmania by Polymerase Chain Reaction and Hybridization with Small Subunit Ribosomal DNA Derived Oligonucleotides

ABSTRACT. A method for discriminating among Leishmania is described, based upon small subunit ribosomal DNA sequence differences. The method was to amplify the entire 2.2 kb small subunit rDNA by polymerase chain reaction using conserved primers specific for the 5′ and 3′ termini of the small subunit ribosomal RNA, and then hybridize the product dotted onto nylon membranes with labeled oligonucleotides. The design of the hybridization probes was based upon complete small subunit rDNA sequences from L. amazonensis, L. major and L. guyanensis and partial sequences of L. mexicana, L. braziliensis, L. tropica and L. chagasi. A high degree of sequence similarity (> 99%) among species was found. However, sufficient sequence divergence occurred to permit the design of internal oligonucleotide probes specific for species complexes. This procedure successfully discriminated amongst a wide range of Leishmania isolates. The method detected as few as 10 cultured organisms and detected parasites in tissue samples from experimentally infected animals. Non‐radioactive labeling showed the same specificity and sensitivity as radioactive probes.

Leishmania tarentolae taxonomic relatedness inferred from phylogenetic analysis of the small subunit ribosomal RNA gene

The sequence of the Leishmania tarentolae SSU rRNA (small subunit or 18S rRNA) gene was completely determined from 2 different strains and used to determine phylogenetic relationships between this organism and other trypanosomatids. Extensive structural similarities were observed between L. tarentolae and mammalian leishmanias the SSU rRNA. Phylogenetic reconstructions, using distance matrix or parsimony methods, showed large evolutionary distances between trypanosomes, either African and American, and L. tarentolae. Further analysis using intergenic rDNA spacer (IGS) sequences as probes in dot blot experiments confirmed the results obtained with the SSU rDNA comparisons. The data presented here clearly indicate that L. tarentolae is closely related to the mammalian parasite Leishmania donovani and highly divergent from trypanosomes.

DNA Sequence Analysis of the Genetic Structure of Populations of Salmonella Enterica and Escherichia Coli

The goal of bacterial population genetics is to understand the factors that determine genetic structure and mediate evolutionary change in natural populations. This broad objective transcends the practical needs of microbiologists for methods of species identification and strain discrimination for epidemiological and other purposes, but the findings of population genetics research have important implications for several branches of medical microbiology (Selander and Musser, 1990), as well as for bacterial systematics.

Forensic Mitochondrial DNA Analysis of 116 Casework Skeletal Samples

ABSTRACT: Between February 1999 and May 2005, 116 DNA extractions were completed on skeletal remains from routine casework. Overall, at least a partial mitochondrial DNA (mtDNA) profile was obtained on 83.6% of samples. Skeletal remains fell into two general categories: (1) samples for body identifications submitted by law enforcement and (2) samples submitted to answer historical or family identity questions. Body identification cases were more likely to yield full mtDNA profiles, whereas historical cases were more likely to result in partial profiles. Overall, the ability to obtain a full or partial profile primarily reflects the difference in the average age and condition of the samples in these two categories and thus, difference in the quantity and quality of the DNA. Cremated remains were uniformly unsuccessful, whereas infant/fetal remains were uniformly successful. Heteroplasmy in skeletal remains was observed at a rate similar to that in hair (∼10%). For body identification cases, skeletal remains had the same mtDNA profile as the accompanying reference sample in 50% of cases.

Commentary on: Divne A-M, Nilsson M, Calloway C, Reynolds R, Erlich H, Allen M. Forensic Casework Analysis Using the HVI/HVII mtDNA Linear Array Assay. J Forensic Sci 2005;50:548–54.

Sir: We read with interest Divne et al.’s ‘‘Forensic casework analysis using the HVI/HVII mitochondrial DNA (mtDNA) linear array assay’’ (1). As caseworking mtDNA forensic examiners with hundreds of mtDNA cases behind us, including experience with hundreds of biological specimens of all kinds (2–4), we urge caution in the use of any mtDNA screening method like the linear array assay that develops only a partial profile on evidentiary material. One of us (T. M.) also has had extensive experience with the precursor technology of linear arrays, SSO typing [see, for example, (5)] and has a good idea of its limitations, especially the potential for a high frequency of ‘‘null’’ or ‘‘blank’’ results caused by polymorphisms that block hybridization, a problem which is barely mentioned by Divne et al. Our forensic concerns are in these general areas: