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Biomedical subjects

Sandra C Smole

Publications and source records attributed to Sandra C Smole.

7 recordsLinked to original sources
PubMed2006

Second generation subtyping: a proposed PulseNet protocol for multiple-locus variable-number tandem repeat analysis of Shiga toxin-producing Escherichia coli O157 (STEC O157).

Most bacterial genomes contain tandem duplications of short DNA sequences, termed "variable-number tandem repeats" (VNTR). A subtyping method targeting these repeats, multiple-locus VNTR analysis (MLVA), has emerged as a powerful tool for characterization of clonal organisms such as Shiga toxin-producing Escherichia coli O157 (STEC O157). We modified and optimized a recently published MLVA scheme targeting 29 polymorphic VNTR regions of STEC O157 to render it suitable for routine use by public health laboratories that participate in PulseNet, the national and international molecular subtyping network for foodborne disease surveillance. Nine VNTR loci were included in the final protocol. They were amplified in three PCR reactions, after which the PCR products were sized using capillary electrophoresis. Two hundred geographically diverse, sporadic and outbreak- related STEC O157 isolates were characterized by MLVA and the results were compared with data obtained by pulsed-field gel electrophoresis (PFGE) using XbaI macrorestriction of genomic DNA. A total of 139 unique XbaI PFGE patterns and 162 MLVA types were identified. A subset of 100 isolates characterized by both XbaI and BlnI macrorestriction had 62 unique PFGE and MLVA types. Although the clustering of isolates by the two subtyping systems was generally in agreement, some discrepancies were observed. Importantly, MLVA was able to discriminate among some epidemiologically unrelated isolates which were indistinguishable by PFGE. However, among strains from three of the eight outbreaks included in the study, two single locus MLVA variants and one double locus variant were detected among epidemiologically implicated isolates that were indistinguishable by PFGE. Conversely, in three other outbreaks, isolates that were indistinguishable by MLVA displayed multiple PFGE types. An additional more extensive multi-laboratory validation of the MLVA protocol is in progress in order to address critical issues such as establishing epidemiologically relevant interpretation guidelines for the MLVA data.

Base Sequence↗
PubMed2004

Identification of a novel peptidoglycan hydrolase CwlM in Mycobacterium tuberculosis.

Mycobacterium tuberculosis is a major global pathogen whose threat has increased with the emergence of multidrug-resistant strains. The cell wall of M. tuberculosis is thick, rigid, and hydrophobic, which serves to protect the organism from the environment and makes it highly impermeable to conventional antimicrobial agents. There is little known about cell wall autolysins (also referred to as peptidoglycan hydrolases) of mycobacteria. We identified an open reading frame (Rv3915) in the M. tuberculosis genome designated cwlM that appeared consistent with a peptidoglycan hydrolase. The 1218-bp gene was amplified by PCR, cloned and expressed in E. coli strain HMS174(DE-3), and its gene product, a 47-kDa recombinant protein, was purified and partially characterized. Purified CwlM was able to lyse whole mycobacteria, release peptidoglycan from the cell wall of Micrococcus luteus and Mycobacterium smegmatis, and cleave N-acetylmuramoyl-L-alanyl-D-isoglutamine, releasing free N-acetylmuramic acid. These results indicate that CwlM is a novel autolysin and identify cwlM as the first, to our knowledge, autolysin gene identified and cloned from M. tuberculosis. CwlM offers a new target for a unique class of drugs that could alter the permeability of the mycobacterial cell wall and enhance the effectiveness of treatments for tuberculosis.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed2004

Selective discrimination of Listeria monocytogenes epidemic strains by a mixed-genome DNA microarray compared to discrimination by pulsed-field gel electrophoresis, ribotyping, and multilocus sequence typing.

Listeria monocytogenes can cause serious illness in humans, and subsequent epidemiological investigation requires molecular characterization to allow the identification of specific isolates. L. monocytogenes is usually characterized by serotyping and is subtyped by using pulsed-field gel electrophoresis (PFGE) or ribotyping. DNA microarrays provide an alternative means to resolve genetic differences among isolates, and unlike PFGE and ribotyping, microarrays can be used to identify specific genes associated with strains of interest. Twenty strains of L. monocytogenes representing six serovars were used to generate a shotgun library, and subsequently a 629-probe microarray was constructed by using features that included only potentially polymorphic gene probe sequences. Fifty-two strains of L. monocytogenes were genotyped by using the condensed array, including strains associated with five major listeriosis epidemics. Cluster analysis of the microarray data grouped strains according to phylogenetic lineage and serotype. Most epidemiologically linked strains were grouped together, and subtyping resolution was the same as that with PFGE (using AscI and ApaI) and better than that with multilocus sequence typing (using six housekeeping genes) and ribotyping. Additionally, a majority of epidemic strains were grouped together within phylogenetic Division I. This epidemic cluster was clearly distinct from the two other Division I clusters, which encompassed primarily sporadic and environmental strains. Discriminant function analysis allowed identification of 22 probes from the mixed-genome array that distinguish serotypes and subtypes, including several potential markers that were distinct for the epidemic cluster. Many of the subtype-specific genes encode proteins that likely confer survival advantages in the environment and/or host.

Bacterial Proteins↗
PubMed2003

Characterization of Mycobacterium montefiorense sp. nov., a novel pathogenic Mycobacterium from moray eels that is related to Mycobacterium triplex.

The characterization of a novel Mycobacterium sp. isolated from granulomatous skin lesions of moray eels is reported. Analysis of the hsp65 gene, small-subunit rRNA gene, rRNA spacer region, and phenotypic characteristics demonstrate that this organism is distinct from its closest genetic match, Mycobacterium triplex, and it has been named M. montefiorense sp. nov.

Animals↗
PubMed2002

Sample preparation of Gram-positive bacteria for identification by matrix assisted laser desorption/ionization time-of-flight.

A new sample preparation method was developed for fresh, whole-cell Gram-positive bacteria to be analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI ToF MS). With fresh, whole-cell Gram-negative bacteria of the Enterobacteriaceae family, we had previously achieved spectra consisting of >50 peaks and mass ranges of 2-25 kDa. Because similar spectral quantity could not be achieved for Gram-positive bacteria, using this same protocol, we investigated an alternative approach that focuses on the thick peptidoglycan layer of the cell wall. Gram-positive bacteria were incubated with 0.05-0.5 mg/ml lysozyme for 30 min prior to being analyzed by MALDI ToF MS. Lysozyme is an enzymatically stable, 14-kDa protein that specifically cleaves between peptidoglycan disaccharide subunits. A significant increase in overall number of peaks (>50) in the 2-14 kDa range was observed without interference from the presence of lysozyme. We show that for four different species (Staphylococcus aureus, S. haemolyticus, Streptococcus pyogenes, and S. agalactiae) reproducible subset of peaks were found within spectra from a reference strain and two unrelated clinical isolates. The data suggests that this sample preparation may be useful for increasing the overall number of peaks within spectra for subsequent development of bacterial identification strategies.

Biomarkers↗
PubMed2002

Clinical and epidemiological correlates of genotypes within the Mycobacterium avium complex defined by restriction and sequence analysis of hsp65.

Species identification of isolates of the Mycobacterium avium complex (MAC) remains a difficult task. Although M. avium and Mycobacterium intracellulare can be identified with expensive, commercially available probes, many MAC isolates remain unresolved, including those representing Mycobacterium lentiflavum as well as other potentially undefined species. PCR restriction analysis (PRA) of the hsp65 gene has been proposed as a rapid and inexpensive approach. We applied PRA to 278 MAC isolates, including 126 from blood of human immunodeficiency virus (HIV)-infected patients, 59 from sputum of HIV-negative patients with chronic obstructive pulmonary disease, 88 from environmental sources, and 5 pulmonary isolates from a different study. A total of 15 different PRA patterns were observed. For 27 representative isolates, a 441-bp fragment of the hsp65 gene was sequenced; based on 54 polymorphic sites, 18 different alleles were defined, including 12 alleles not previously reported. Species and phylogenetic relationships were more accurately defined by sequencing than by PRA or commercial probe. The distribution of PRA types and, by implication, phylogenetic lineages among blood isolates was significantly different from that for pulmonary and environmental isolates, suggesting that particular lineages have appreciably greater virulence and invasive potential.

Bacterial Proteins↗