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[Bacterial genotyping in nosocomial infections].

Hospitalized patients are at unusually high risk of infections, and furthermore, the hospital environment favors the acquisition of resistance to antimicrobial agents, complicating the treatment of nosocomial infections due to drug-resistant pathogens. The prevention of nosocomial infections, based on a surveillance system as an essential element of an infection control program, is the only way to reduce morbidity and mortality. A typing method for strain clonality permits the infection control program to confirm the association between infected patients and the reservoir for the microorganisms of interest and to determine modes of transmission, because the mode of transmission or reservoir may not be the same for multiple strains of a bacterial species. An ideal method of subtyping bacterial isolates from a given species should be simple, rapid, sensitive and discriminatory. Traditionally, once bacterial isolates from an outbreak have been determined to be of the same species, further evaluation for similarity or relatedness has been based on phenotypic methods. Biotyping, serotyping, phagetyping, and antibiotype determination are not always adequately sensitive to distinguish unrelated strains with similar phenotypes. Within phenotypic methods, multilocus enzyme electrophoresis is a powerful tool but because of its complexity it is not likely to become widely available for study of local outbreaks of bacterial infections. In recent years, molecular genetic methods, including plasmid profile, genomic restriction fragment length polymorphism analyses by conventional electrophoresis or by pulsed-field gel electrophoresis, single chromosomal gene polymorphism by DNA hybridization or by PCR amplification, ribotyping, and genomic fingerprinting generated by repetitive element sequence-based polymerase chain reaction, have been useful in evaluating endemic infections and outbreaks of a variety of nosocomial pathogens. For epidemiologic studies, genotyping systems based on defined chromosomal genes or whole DNA polymorphism provide significant advantages over plasmid analysis. Among plasmid-non-based genotypic methods, the choice depends on i) the examination of how much discrimination the method can add for the epidemiologic investigation, ii) the resources available to the laboratory, and iii) the level of expertise of the personnel involved in the testing because, until standardized rules of interpretation are published, the same data may be interpreted in different ways by different investigators. Finally, biotyping, serotyping, and antibiotype determination remain an appropriate first step for the evaluation of apparent outbreaks with the caveat that different strains with the same phenotypic properties may exist concurrently within the same environment. Genotyping may be a second level of analysis to evaluate relatedness of bacterial strains, because the use of molecular biology techniques should support an epidemiologic investigation rather than initiate it.

Bacterial Typing Techniques

On the use of DNA fingerprints for linkage studies in cattle.

To find a marker for the bovine "muscular hypertrophy" gene and for the "roan" locus, we have typed six cattle pedigrees totaling 540 animals for nine blood group systems, for 12 biochemical markers, for RFLPs at four loci, and with five probes revealing multilocus DNA fingerprints. Segregation analysis of the fingerprint bands showed that, in cattle, a fingerprint probe will reveal a mean of 7.6 clearly resolvable bands, behaving as simple, highly informative Mendelian entities characterized by a mean mutation rate of +/- 1/4500 gametes. For one of the bands, we observed a "mutation burst" generating germline mosaicism. Because some of the fingerprint bands were allelic or corresponded to clustered minisatellites, a mean of only 5.7 independent loci is explored per probe. Fingerprint bands revealed by different probes also show a clear propensity for close linkage, pointing toward nonrandom distribution of minisatellite sequences or the existence of minisatellite clusters. Although this reduces the power of fingerprints for linkage analysis substantially, we were able to demonstrate genetic linkage between fingerprint bands and at least three of the classical markers, to exclude the roan locus from 4.5 Morgans of the bovine genome with the DNA fingerprints and for an additional 2.5 Morgans with the classical markers, and to identify a solid candidate marker for the bovine muscular hypertrophy gene, yielding a lod score greater than or equal to 2.84 without any obliged recombinant.

Animals

Principles and recent advances in human DNA fingerprinting.

Since 1985, DNA typing systems have played an increasingly important role in many aspects of human genetics, most notably in forensic and legal medicine. This article reviews the development of multilocus and single locus minisatellite DNA probes, and more recently the use of PCR to amplify hypervariable DNA loci, as well as discussing the biological properties of the unstable regions of DNA which form the basis of almost all DNA fingerprinting systems.

Base Sequence

Species limits in Rhizobium populations that nodulate the common bean (Phaseolus vulgaris).

Evolutionary genetic relationships among 146 bean-nodulating Rhizobium strains, including 94 field isolates from three localities in Colombia and 36 strains from Mexico, were examined by multilocus enzyme electrophoresis and restriction fragment length polymorphism analysis of a PCR-amplified 260-bp segment of the 16S rRNA gene. Seventy-five electrophoretic types (ETs), corresponding to multilocus enzyme genotypes, were identified, including a genotypically diverse group of 18 ETs in Colombia that is strongly differentiated from the ETs of R. etli, which occur in Mexico, Colombia, and Brazil. Most strains of the distinctive Colombian ETs carried the same 16S rRNA allele as did strains of R. etli, but, surprisingly, 17 isolates of two of these ETs had the allele that is characteristic of R. leguminosarum, and strains of two other divergent groups of ETs were also polymorphic for the two alleles. No fully satisfactory explanation for the occurrence of the R. leguminosarum 16S rRNA allele in three distantly related groups of strains is available, but horizontal transfer and recombination of the gene, in whole or in part, would seem to be more plausible than convergence in nucleotide sequence.

Alleles

Evolutionary genetics of the encapsulated strains of Haemophilus influenzae.

Genetic relationships among 2209 isolates of Haemophilus influenzae of polysaccharide capsule serotypes a, b, c, d, e, and f were determined by analyzing electrophoretically demonstrable allelic variation at 17 chromosomal enzyme loci. We distinguished 280 electrophoretic types (ETs), representing distinctive multilocus genotypes. Genetic diversity among ETs of isolates of the same serotype was, on average, only 67% of that in the total sample, and no ETs were shared among isolates of different serotypes. Cluster analysis of the ETs revealed 2 primary phylogenetic divisions at a genetic distance of 0.66 and 12 major lineages diverging from one another at distances greater than 0.42. In general, strains of different phylogenetic lines or groups of allied lineages have characteristic cap region restriction fragment length polymorphism patterns obtained by digestion of genomic DNA with EcoRI. Strains producing serotype c, e, and f capsules have no close relationships to those of other encapsulated strains. Lineages of both serotype a and b strains occur in each primary phylogenetic division, most probably as a result of the transfer of serotype-specific sequences of the cap region between clonal lineages. Serotype a strains allied in division I with a group of abundant serotype b clones and the serotype d strains apparently are more virulent than the serotype a strains in division II, which are related to serotype b and f strains that rarely cause invasive disease.

Alleles

Diallelic multilocus model of neutral genes.

The evolution of a completely linked diallelic multilocus system of neutral genes in a finite population is studied. A diffusion model incorporating random genetic drift and mutation is used. We neglect the recombination. To begin with, the spectral analysis of the Kolmogorov backward equation for this model is investigated. We apply this to two extreme situations when the number of sites approaches to infinity. One is a DeMoivre-Laplace type approximation and the other is a Poisson type approximation. The former is applied to the study of the simultaneous distribution and evolution of a large number of neutral genes. It is applicable to the distribution of a polygenic character controlled by clustered loci on a chromosome, and we show that it differs from the normal distribution on account of random genetic drift and linkage disequilibrium. The latter is applied to the distribution of the number of segregating sites in DNA nucleotide sequences, and the rate of evolution is obtained.

Alleles

A recombination event occurring within two complex 5q13.1 microsatellite repeat polymorphisms suggests a telomeric mapping of spinal muscular atrophy.

The gene for the childhood spinal muscular atrophies (SMAs) has been mapped to 5q13.1. The interval containing the SMA gene has been defined by linkage analysis as 5qcen-D5S629-SMA-D5S557-5qter. We have identified a recombination event within this interval on a type-I SMA chromosome. The recombination maps to a region of multilocus microsatellite repeat (MSR) markers, and occurs between different subloci of two such markers, CMS-1 and 7613. While the possibility of a novel mutation caused by the recombination cannot be discounted, we believe when viewed in the context of a similar recombination in a Dutch SMA family, a centromeric boundary at the recombination site for the critical SMA interval is likely. This new proximal boundary would reduce the minimal region harboring the SMA locus from approximately 1.1 Mb to approximately 600 kb.

Alleles

Induction of deletion mutations by methoxyacetaldehyde in Chinese hamster ovary (CHO)-AS52 cells.

We have reported previously that methoxyacetaldehyde (MALD), a metabolite of 2-methoxyethanol, induces gpt gene mutations in Chinese hamster ovary (CHO)-AS52 cells but not hprt gene mutations in the standard CHO-K1-BH4 cells. In addition, MALD induces chromosome aberrations in both CHO cell lines. The data presented suggest that MALD induces deletion-type mutations. In this study, we analyzed MALD-induced CHO-AS52 mutants for deletion-type mutations using the nested-polymerase chain reaction (nested-PCR) assay. Spontaneous CHO-AS52 mutants are used as untreated control. Ethylnitrosourea (ENU)-induced CHO-AS52 mutants are used as negative control for multilocus deletions since ENU is a potent inducer of point mutations. The results show that the frequency of MALD-induced mutants containing total deletion of the gpt gene is 42.4% which is 2.3-fold higher than that from spontaneous mutants (18.6%). The frequency of ENU-induced deletion mutation is 3%. The data substantiate our hypothesis that MALD induces major deletion mutations.

Acetaldehyde

Clonal relationships among Escherichia coli strains that cause hemorrhagic colitis and infantile diarrhea.

The genetic relationships among 1,300 isolates of Escherichia coli representing 16 serotypes associated with enteric disease, including O157:H7 strains recovered from patients with hemorrhagic colitis and hemolytic uremic syndrome and O26:H11, O55:H6, O55:H7, O111:H2, and O128:H2 strains, many of which were isolated originally from infants with diarrhea, were estimated from allelic variation among 20 enzyme-encoding genes detected by multilocus enzyme electrophoresis. Multiple electrophoretic types were observed among isolates of each serotype, with isolates of the same O serogroup differing on average at 28% of the enzyme loci. Comparisons of the multilocus enzyme profiles revealed that 72% of the isolates belong to 15 major electrophoretic types, each of which corresponds to a bacterial clone with a wide geographic distribution. Genetically, the O157:H7 clone is most closely related to a clone of O55:H7 strains that has long been associated with worldwide outbreaks of infantile diarrhea. We propose that the new pathogen emerged when an O55:H7-like progenitor, already possessing a mechanism for adherence to intestinal cells, acquired secondary virulence factors (Shiga-like cytotoxins and plasmid-encoded adhesins) via horizontal transfer and recombination.

Alleles

Oligonucleotide probes to the 16S ribosomal RNA: implications of sequence homology and secondary structure with particular reference to the oral species Prevotella intermedia and Prevotella nigrescens.

Eight oligonucleotides based upon regions of the small subunit 16S ribosomal RNA gene sequences were analysed against a background of their position within the molecule and their two-dimensional structure to rationalise their use in recognising Prevotella intermedia and Prevotella nigrescens. The 41 clinical isolates from both oral and respiratory sites and two reference strains were subjected to DNA-DNA hybridisation and multilocus enzyme electrophoresis to confirm their identity. Alignment of oligonucleotide probes designated I Bi-2 to I Bi-6 (for P. intermedia) and 2Bi-2 (for P. nigrescens) with the 16S rRNA suggested that these probes lacked specificity or were constructed from hypervariable regions. A 52-mer oligonucleotide (designated Bi) reliably detected both species. Because of the high degree of concordance between the 16S rRNAs of both species, it was necessary to vary the stringency of hybridisation conditions for detection of both species. Thus probe I Bi-I recognised P. intermedia while I Bi-I detected both P. intermedia and P. nigrescens at low stringency. However, under conditions of high stringency only P. nigrescens was recognised by probe 2Bi-I. These probes were highly specific and did not hybridise with DNA from the closely related P. corporis, nor other periodontal pathogens such as Fusobacterium nucleatum, Actinobacillus actinomycetemcomitans, Treponema denticola and several pigmented species such as Prevotella melaninogenica, P. denticola, P. loescheii, Porphyromonas asaccharolytica, Py. endodontalis, Py. gingivalis, Py. levii, and Py. macacae.

Bacterial Typing Techniques

DNA sequence analysis of X-ray induced Adh null mutations in Drosophila melanogaster.

The mutational spectrum for 28 X-ray induced mutations and 2 spontaneous mutations, previously determined by genetic and cytogenetic methods, consisted of 20 multilocus deficiencies (19 induced and 1 spontaneous) and 10 intragenic mutations (9 induced and 1 spontaneous). One of the X-ray induced intragenic mutations was lost, and another was determined to be a recombinant with the allele used in the recovery scheme. The DNA sequence of two X-ray induced intragenic mutations has been published. This paper reports the results of DNA sequence analysis of the remaining intragenic mutations and a summary of the X-ray induced mutational spectrum. Only one of the X-ray induced mutations is a single base change, a C to G transversion (AdhnLA80). Therefore, the mutational spectrum of X-ray induced mutations consists predominantly of deletions that are observed to range in size from two base pairs to deletions of a large number of loci as determined by genetic complementation analysis. The combination of DNA sequence analysis with genetic complementation analysis shows a continuous distribution in size of deletions rather than two different types of mutations consisting of deletions and "point mutations." Sequencing is shown to be essential for detecting intragenic deletions. Of particular importance for future studies is the observation that all of the intragenic deletions consist of a direct repeat adjacent to the break-point with one of the repeats deleted.

Alcohol Dehydrogenase

Allelic variation in human mitochondrial genes based on patterns of restriction site polymorphism.

Restriction maps of 145 human mtDNAs representing samples from five geographic regions were used to construct multilocus genotypes for 28 genetic loci of the mitochondrial genome. Alleles were defined as distinct combinations of the presence or absence of polymorphic restriction sites within each locus. The 28 loci included 13 genes encoding proteins, 10 genes specifying tRNAs, 2 genes specifying rRNAs, and 3 noncoding regions consisting of the D loop, the light strand origin of replication, and the 5' noncoding sequence. In 35 comparisons of allele frequency distributions to expected distributions predicted by neutral mutation theory (assuming an infinite alleles model), the results revealed that most genetic diversity values (71%) fell within the range predicted by the neutral model; however, excesses in the frequencies of common alleles and in the number of singleton alleles within populations were observed at specific loci. Departures from the neutral mutation model are most readily explained by the effects of the recent expansion of the human population and the action of purifying selection. Coefficients of population differentiation suggest that gene flow of mtDNA types between certain geographic regions may be limited.

Alleles

Genetic differences at four DNA typing loci in Finnish, Italian, and mixed Caucasian populations.

Highly polymorphic segments of the human genome containing variable numbers of tandem repeats (VNTRs) have been widely used to establish DNA profiles of individuals for use in forensics. Methods of estimating the probability of occurrence of matching DNA profiles between two randomly selected individuals have been subject to extensive debate regarding the possibility of significant substructure occurring within the major races. We have sampled two Caucasian subpopulations, Finns and Italians, at four commonly used VNTR loci to determine the extent to which the subgroups differ from each other and from a mixed Caucasian database. The data were also analyzed for the occurrence of linkage disequilibrium among the loci. The allele frequency distributions of some loci were found to differ significantly among the subpopulations in a manner consistent with population substructure. Major differences were also found in the probability of occurrence of matching DNA profiles between two individuals chosen at random from the same subpopulation. With respect to the Finnish and Italian subpopulations, the conventional product rule for estimating the probability of a multilocus VNTR match using a mixed Caucasian database consistently yields estimates that are artificially small. Systematic errors of this type were not found using the interim ceiling principle recently advocated in the National Research Council's report [National Research Council (1992) DNA Technology in Forensic Science (Natl. Acad. Sci., Washington)]. The interim ceiling principle is based on currently available racial or ethnic databases and sets an arbitrary lower limit on each VNTR allele frequency. In the future the ceiling frequencies are expected to be established from more adequate data acquired for relevant VNTR loci from multiple subpopulations.

Alleles

Genetic structure of a lotic population of Burkolderia (Pseudomonas) cepacia.

The genetic structure of a population of Burkholderia (Pseudomonas) cepacia isolated from a southeastern blackwater stream was investigated by using multilocus enzyme electrophoresis to examine the allelic variation in eight structural gene loci. Overall, 213 isolates were collected at transect points along the stream continuum, from both the sediments along the bank and the water column. Multilocus enzyme electrophoresis analysis revealed 164 distinct electrophoretic types, and the mean genetic diversity of the entire population was 0.574. Genetic diversity values did not vary spatially along the stream continuum. From a canonical discriminant analysis, Mahalonobis distances (measurements of genetic similarity between populations) revealed significant differences among the subpopulations at the sediment sampling points, suggesting bacterial adaptation to a heterogeneous (or patchy) microgeographical environment. Multilocus linkage disequilibrium analysis of the isolates revealed only limited association between alleles, suggesting frequent recombination, relative to binary fission, in this population. Furthermore, the dendrogram created from the data of this study and the allele mismatch distribution are typical of a population characterized by extensive genetic mixing. We suggest that B. cepacia be added to the growing list of bacteria that are not obligatorily clonal.

Alleles

Toxoplasma gondii comprises three clonal lineages: correlation of parasite genotype with human disease.

The population genetic structure of Toxoplasma gondii was determined by multilocus restriction fragment length polymorphism analysis at 6 loci in 106 independent isolates from humans and animals. Phylogenetic and statistical analyses indicated a highly unusual population structure consisting of 3 widespread clonal lineages. Extensively mixed genotypes were only apparent in 4 strains, which indicated that, while not separate species, sexual recombination between the 3 lineages is exceedingly rare in natural populations. T. gondii is a major cause of subclinical human infection and an important opportunistic pathogen that causes severe disease in immunocompromised patients. While strains from all 3 lineages were isolated from humans, the majority of human toxoplasmosis cases were associated with strains of a type II genotype. The correlation of specific clonal lineages with human toxoplasmosis has important implications for development of vaccines, drug treatments, and diagnostic protocols.

Animals

The nature of X-ray-induced mutations in mature sperm and spermatogonial cells of Drosophila melanogaster.

Mutations at four X-linked visible loci (yellow, white, vermilion and forked) induced by X-irradiation of mature sperm and spermatogonial cells were analysed genetically and cytogenetically. In addition, a fraction of the intragenic vermilion mutations was analysed molecularly. Males of two wild-type strains (Amherst M56i and Berlin-K) were used. A total of 332,651 chromosomes of irradiated mature sperm and 311,567 of irradiated spermatogonial cells were scored. The ratio of F1 female sterile, F2 male lethal and F2 male viable mutations in mature sperm and spermatogonial cells is very similar. The cytogenetic analysis shows equal fractions of multilocus deletions and translocations among the mutations recovered from both stages of spermatogenesis. These data strongly suggest that the spectrum of X-ray mutations is similar in mature sperm and spermatogonial cells, including multilocus deletions and chromosome rearrangements. The molecular analysis of a number of intragenic vermilion mutations showed the presence of three small deletions (1-10 bp), one insertion of two nucleotides and seven single nucleotide changes.

Animals