O Olsvik

Magnetic separation techniques in diagnostic microbiology.

The principles of magnetic separation aided by antibodies or other specific binding molecules have been used for isolation of specific viable whole organisms, antigens, or nucleic acids. Whereas growth on selective media may be helpful in isolation of a certain bacterial species, immunomagnetic separation (IMS) technology can isolate strains possessing specific and characteristic surface antigens. Further separation, cultivation, and identification of the isolate can be performed by traditional biochemical, immunologic, or molecular methods. PCR can be used for amplification and identification of genes of diagnostic importance for a target organism. The combination of IMS and PCR reduces the assay time to several hours while increasing both specificity and sensitivity. Use of streptavidin-coated magnetic beads for separation of amplified DNA fragments, containing both biotin and a signal molecule, has allowed for the conversion of the traditional PCR into an easy-to-read microtiter plate format. The bead-bound PCR amplicons can also easily be sequenced in an automated DNA sequencer. The latter technique makes it possible to obtain sequence data of 300 to 600 bases from 20 to 30 strains, starting with clinical samples, within 12 to 24 h. Sequence data can be used for both diagnostic and epidemiologic purposes. IMS has been demonstrated to be a useful method in diagnostic microbiology. Most recent publications describe IMS as a method for enhancing the specificity and sensitivity of other detection systems, such as PCR, and providing considerable savings in time compared with traditional diagnostic systems. The relevance to clinical diagnosis has, however, not yet been fully established for all of these new test principles. In the case of PCR, for example, the presence of specific DNA in a food sample does not demonstrate the presence of a live organism capable of inducing a disease. However, all tests offering increased sensitivity and specificity of detection, combined with reduced time of analysis, have to be seriously evaluated. Images

A nested PCR followed by magnetic separation of amplified fragments for detection of Escherichia coli Shiga-like toxin genes

The Shiga-like toxin (SLT) I and II genes in cytotoxic Escherichia coli strains were detected using a polymerase chain reaction (PCR) procedure. Identification and differentiation of SLT I and II was carried out using primers giving PCR-generated DNA fragments of different size for the two cytotoxins. A two-step PCR procedure utilizing three primers in a nested configuration for both SLT I and II was combined with magnetic separation to identify the toxin genes in a rapid, specific and sensitive test system designated DIANA (Detection of Immobilized Amplified Nucleic Acid). The first PCR was carried out using standard methods, and the product generated was used as primer in the second PCR. In this procedure one of the primers from the first PCR was used with biotin label, and the second (inner) primer was 32P-labelled. The double-stranded DNA fragments generated containing the two primers, were biotinylated on one 5 end and 32P-labelled on the other 5 end. These fragments were separated from the solution using streptavidin-coated super-paramagnetic microscopic beads. The test could detect and differentiate between SLT I and II in a positive/negative ratio of more than 20. The assay could detect five SLT-positive E. coli organisms in the 5 microliters test sample. The presence of 100-fold more SLT-negative strains in a sample did not adversely affect the test signal.

Antimicrobial resistance of enteropathogenic Escherichia coli strains from a nosocomial outbreak in Kenya

The majority of the 78 enteropathogenic (EPEC) and the 151 non‐EPEC Escherichia coli strains isolated from preterm neonates during an outbreak of gastroenteritis in a hospital in Nairobi, Kenya, were resistant to trimethoprim‐sulfamethoxaxole, chloramphenicol, oxytetracycline and ampicillin, but only a few strains were resistant to cefazolin, cefamandole, cefotaxime, amikacin and nalidixic acid. Fourteen different antimicrobial resistance patterns were observed in the 229 strains of E. coli analysed. Eighty‐two percent of the EPEC strains belonged to two resistance patterns compared with 79% of non‐EPEC strains which exhibited three resistance patterns. There was no consistent relationship between plasmid profile group and antimicrobial resistance pattern, although one resistance pattern was more frequently observed in EAF‐positive strains belonging to the dominant plasmid profile group. Nine percent of the EPEC strains were resistant to gentamicin compared to 37% in the non‐EPEC group. No correlation was observed between administration of gentamicin and percentage of resistant strains isolated. None of the nine neonates receiving gentamicin died during the outbreak. Gentamicin resistance was observed in E. coli strains from six out of these nine neonates. Five out of fourteen neonates who received other antimicrobials, or no antibiotic treatment at all, died.

Cholera in the Americas: Foodborne Aspects

Over 100 serotypes of Vibrio cholerae exist, but generally the toxigenic strains of the serogroup O1 cause cholera and possess documented epidemic potential. The main symptom of cholera is a profuse diarrhea resulting in dehydration, that if untreated, leads to death. Seven pandemics of this contagious disease have been recorded during the last 200 years. A sick person secrets in his stool billions of organisms daily, and water and food contaminated with such a stool are the primary sources of infection during the epidemics. With the increase of the international food trade, food is often shipped from countries with endemic or epidemic cholera. With one exception, no documented cases of cholera have been reported, as a result of the internationally regulated food trade. However, during the present Latin American epidemic, inadequately cooked seafood has been implicated as a source of cholera. As a result of the epidemic, over 100 cases of cholera have occurred in the United States related to seafood consumed during a visit to Latin America or after its noncommercial transport into the country. Furthermore, V. cholerae persists as a free-living organism in environmental reservoirs in Australia and the U.S. Gulf Coast; there have been 65 domestically acquired cases of cholera in the United States since 1973. Molecular typing methods have enabled us to identify and characterize endemic and epidemic strains, and to document transmission of cholera when food was implicated epidemiologically as a vehicle of transmission.

Molecular Characterization of Corynebacterium diphtheriae Isolates, Russia, 1957-1987

In the 1990s, the Newly Independent and Baltic States of the former Soviet Union experienced the largest diphtheria outbreak since the 1960s; it was caused by Corynebacterium diphtheriae strains of a unique clonal group. To address its origin, we studied 47 clinical isolates from Russia and demonstrated that this clonal group was an integral part of the endemic reservoir that existed in Russia at least 5 years before the epidemic began.

Tetracycline resistance genes in Kenyan hospital isolates of Salmonella typhimurium

All 97 strains of Salmonella typhimurium isolated from patients at a hospital in Nairobi, Kenya, during 1988‐90 were resistant to tetracycline. The minimum inhibitory concentration (MIC) showed a large distribution range from 1 μg/ml to 128 μg/ml. The strains were heterogeneous with respect to plasmid content, but initially all strains possessed, in addition to other plasmids, a large 60‐, 63‐or 65‐MDa plasmid. The tetracycline resistance genes were characterized using oligonucleotide probes, and 20% of the resistant strains possessed tetracycline type A (tetrA), 6%tetr B, and 4%tetrC genes. Three strains possessed both type A and B tetracycline resistance determinants, which were shown to be located on the large 65‐MDa plasmid. There was no correlation between strains isolated from stools, blood, cerebrospinal or epidural fluids, pus, or urine, with respect to the tetracycline genotypes, MIC values or plasmid content.

Detection of Virulence Determinants in Enteric Escherichia coli Using Nucleic Acid Probes and Polymerase Chain Reaction

As certain strains of Escherichia coli are a major cause of diarrhea both in man and animals, diagnosis of the specific etiological agents must be carried out beyond the species level. Several different pathogroups have been identified and for some of them, also the specific virulence determinants. Traditional microbiological assays have to a certain degree been supplemented or replaced with nucleic acid-based methods, like hybridization assays using cloned fragments or oligonucleotides as probes. Recently, the polymerase chain reaction (PCR) has been shown to be a suitable tool for differentiation between E. coli strains belonging to the normal enteric flora, and those carrying specific virulence determinants. Probe assays have been established in some diagnostic laboratories and found to be reliable for detection of the different enterotoxins of both the heat-stable-(ST) and the heat-labile-(LT) -families, and the shiga-like cytotoxins (SLTs). Different setups of the PCR principle have also been used to identify and characterize such genes. Genes encoding some of the important adhesion fimbria, characterizing such toxin-producing strains, have been targets for some assays. Several of the genes involved in the invasion process of the enteroinvasive E. coli are routinely identified using probe assays, avoiding the use of cell-cultures or laboratory animals. The enteropathogenic E. coli strains have for years been defined as a serological cluster, and different probes for both plasmidial and chromosomal genes have now confirmed the presence of common virulence properties. Probes and PCR are becoming very important diagnostic tools in identification and characterization of virulence- or virulence-associated genes in E. coli.

Immunoaffinity Separation of Cells Using Monosized Magnetic Polymer Beads

A widespread biomedical use of magnetic particles has recently been initiated as a result of the preparation of a new generation of magnetic particles by Ugelstad et al. (1980). Originally, these particles were used for removal of tumor cells from bone marrow, and selective isolation of cells remains a very important area of application. More recently, their area of application has been extended to include prokaryotic cells, viruses, and subcellular components. A new and rapidly increasing area of application for magnetic beads is in different fields of DNA technology; this is discussed in Chapter 31 of this volume.

A unique plasmid profile characterizing Salmonella enteritidis isolates from patients and employees in a hospital.

Plasmid profiling was used as an epidemiological tool during a period of frequent Salmonella enteritidis infection in a hospital. S. enteritidis was isolated from 22 patients and employees. Isolates from 18 persons harbored one 29 and one 36 megadalton (MDa) plasmid. The 29 MDa plasmid has not been previously described in this species and was not found in 54 control strains of S. enteritidis from other sources. The respective restriction endonuclease digest fragments of the 36 and the 29 MDa plasmids were always identical. This plasmid pattern thus served as a marker for the isolates from the outbreak.

Affinity Separation of Nucleic Acids on Monosized Magnetic Beads

The introduction of solid-phase methods has had great impact on molecular biology in the area of synthesis and sequencing of proteins and DNA. High yield and reproducibility can be obtained, and automation is facilitated since reaction buffers can be rapidly and easily changed. There are many alternative solid supports, such as organic or inorganic polymer beads or the walls of test tubes or microtiter wells. An attractive alternative is the use of monosized magnetic particles, Dynabeads (Lea et al., 1988; Ugelstad et al., this volume, Chapter 16), which combines the convenience of magnetic motility with an adsorption and desorption of biomolecules at the surface. This provides reaction kinetics similar to those found in a free solution. Magnetic beads with covalently bound streptavidin can be used for directed immobilization of both double-stranded and single-stranded biotinylated DNA. The remarkable stability and strength of the biotin-streptavidin interaction (K d = 10−15 M) allow DNA manipulations, such as strand melting, elution, and hybridization (using alkali, temperature, or formamide) to be performed without interfering with the binding of the DNA to the beads.