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Comparison of traditional and molecular methods of typing isolates of Staphylococcus aureus.

Fifty-nine Staphylococcus aureus isolates and 1 isolate of Staphylococcus intermedius were typed by investigators at eight institutions by using either antibiograms, bacteriophage typing, biotyping, immunoblotting, insertion sequence typing with IS257/431, multilocus enzyme electrophoresis, restriction analysis of plasmid DNA, pulsed-field or field inversion gel electrophoresis, restriction analysis of PCR-amplified coagulase gene sequences, restriction fragment length polymorphism typing by using four staphylococcal genes as probes, or ribotyping. Isolates from four well-characterized outbreaks (n = 29) and a collection of organisms from two nursing homes were mixed with epidemiologically unrelated stock strains from the Centers for Disease Control and Prevention. Several isolates were included multiple times either within or between the sets of isolates to analyze the reproducibilities of the typing systems. Overall, the DNA-based techniques and immunoblotting were most effective in grouping outbreak-related strains, recognizing 27 to 29 of the 29 outbreak-related strains; however, they also tended to include 3 to 8 epidemiologically unrelated isolates in the same strain type. Restriction fragment length polymorphism methods with mec gene-associated loci were less useful than other techniques for typing oxacillin-susceptible isolates. Phage typing, plasmid DNA restriction analysis, and antibiogram analysis, the techniques most readily available to clinical laboratories, identified 23 to 26 of 29 outbreak-related isolates and assigned 0 to 6 unrelated isolates to outbreak strain types. No single technique was clearly superior to the others; however, biotyping, because it produced so many subtypes, did not effectively group outbreak-related strains of S. aureus.

Bacterial Typing Techniques↗

Identification of a Streptococcus agalactiae serotype III subtype 4 clone in association with adult invasive disease in Hong Kong.

Characterization of Streptococcus agalactiae serotype III isolates revealed a subtype 4 clone that has an indistinguishable pulsed-field gel electrophoresis pattern and possesses a C-alpha protein, IS1381, and a novel sequence type (ST), ST 283, by multilocus sequence tagging. This clone was significantly associated with diseases caused by invasive strains from nonpregnant adults (P <or= 0.01, chi-square test) and was not present in the genital tracts of pregnant mothers.

DNA Transposable Elements↗

Molecular evolution and host adaptation of Bordetella spp.: phylogenetic analysis using multilocus enzyme electrophoresis and typing with three insertion sequences.

A total of 188 Bordetella strains were characterized by the electrophoretic mobilities of 15 metabolic enzymes and the distribution and variation in positions and copy numbers of three insertion sequences (IS). The presence or absence of IS elements within certain lineages was congruent with estimates of overall genetic relationships as revealed by multilocus enzyme electrophoresis. Bordetella pertussis and ovine B. parapertussis each formed separate clusters, while human B. parapertussis was most closely related to IS1001-containing B. bronchiseptica isolates. The results of the analysis provide support for the hypothesis that the population structure of Bordetella is predominantly clonal, with relatively little effective horizontal gene flow. Only a few examples of putative recombinational exchange of an IS element were detected. Based on the results of this study, we tried to reconstruct the evolutionary history of different host-adapted lineages.

Adaptation, Biological↗

Application of molecular techniques to the study of hospital infection.

Nosocomial infections are an important source of morbidity and mortality in hospital settings, afflicting an estimated 2 million patients in United States each year. This number represents up to 5% of hospitalized patients and results in an estimated 88,000 deaths and 4.5 billion dollars in excess health care costs. Increasingly, hospital-acquired infections with multidrug-resistant pathogens represent a major problem in patients. Understanding pathogen relatedness is essential for determining the epidemiology of nosocomial infections and aiding in the design of rational pathogen control methods. The role of pathogen typing is to determine whether epidemiologically related isolates are also genetically related. To determine molecular relatedness of isolates for epidemiologic investigation, new technologies based on DNA, or molecular analysis, are methods of choice. These DNA-based molecular methodologies include pulsed-field gel electrophoresis (PFGE), PCR-based typing methods, and multilocus sequence analysis. Establishing clonality of pathogens can aid in the identification of the source (environmental or personnel) of organisms, distinguish infectious from noninfectious strains, and distinguish relapse from reinfection. The integration of molecular typing with conventional hospital epidemiologic surveillance has been proven to be cost-effective due to the associated reduction in the number of nosocomial infections. Cost-effectiveness is maximized through the collaboration of the laboratory, through epidemiologic typing, and the infection control department during epidemiologic investigations.

Bacteria↗

Clonal relationship of recent invasive Haemophilus influenzae serotype f isolates from Denmark and the United States.

Surveillance performed after the introduction of general Haemophilus influenzae serotype b (Hib) vaccination in Denmark identified 13 cases of invasive bacteraemic H. influenzae serotype f (Hif) disease in adults over a period of 7 years. Bacteraemic respiratory tract infections accounted for 61 % of cases, but meningitis, epiglottitis and osteoarthritis were also seen. Recent Danish isolates were compared to recent American isolates, historical Hif strains and non-Hif invasive strains. Results of conventional serotyping were confirmed by PCR detection of the serotype-f-specific cap and bexA gene sequences. Multilocus enzyme electrophoresis typing revealed that recent Danish and American isolates belonged to a single Hif clone, which may be undergoing expansion. The need for accurate serotyping of H. influenzae to enable reliable monitoring for Hib replacement by other capsular types is emphasized.

ATP-Binding Cassette Transporters↗

Similarity and differences in the Lactobacillus acidophilus group identified by polyphasic analysis and comparative genomics.

A set of lactobacilli were investigated by polyphasic analysis. Multilocus sequence analysis, DNA typing, microarray analysis, and in silico whole-genome alignments provided a remarkably consistent pattern of similarity within the Lactobacillus acidophilus complex. On microarray analysis, 17 and 5% of the genes from Lactobacillus johnsonii strain NCC533 represented variable and strain-specific genes, respectively, when tested against four independent isolates of L. johnsonii. When projected on the NCC533 genome map, about 10 large clusters of variable genes were identified, and they were enriched around the terminus of replication. A quarter of the variable genes and two-thirds of the strain-specific genes were associated with mobile DNA. Signatures for horizontal gene transfer and modular evolution were found in prophages and in DNA from the exopolysaccharide biosynthesis cluster. On microarray hybridizations, Lactobacillus gasseri strains showed a shift to significantly lower fluorescence intensities than the L. johnsonii test strains, and only genes encoding very conserved cellular functions from L. acidophilus hybridized to the L. johnsonii array. In-silico comparative genomics showed extensive protein sequence similarity and genome synteny of L. johnsonii with L. gasseri, L. acidophilus, and Lactobacillus delbrueckii; moderate synteny with Lactobacillus casei; and scattered X-type sharing of protein sequence identity with the other sequenced lactobacilli. The observation of a stepwise decrease in similarity between the members of the L. acidophilus group suggests a strong element of vertical evolution in a natural phylogenetic group. Modern whole-genome-based techniques are thus a useful adjunct to the clarification of taxonomical relationships in problematic bacterial groups.

Genetic Variation↗

Molecular epidemiologic typing systems of bacterial pathogens: current issues and perspectives.

The epidemiologic typing of bacterial pathogens can be applied to answer a number of different questions: in case of outbreak, what is the extent and mode of transmission of epidemic clone(s)? In case of long-term surveillance, what is the prevalence over time and the geographic spread of epidemic and endemic clones in the population? A number of molecular typing methods can be used to classify bacteria based on genomic diversity into groups of closely-related isolates (presumed to arise from a common ancestor in the same chain of transmission) and divergent, epidemiologically-unrelated isolates (arising from independent sources of infection). Ribotyping, IS-RFLP fingerprinting, macrorestriction analysis of chromosomal DNA and PCR-fingerprinting using arbitrary sequence or repeat element primers are useful methods for outbreak investigations and regional surveillance. Library typing systems based on multilocus sequence-based analysis and strain-specific probe hybridization schemes are in development for the international surveillance of major pathogens like Mycobacterium tuberculosis. Accurate epidemiological interpretation of data obtained with molecular typing systems still requires additional research on the evolution rate of polymorphic loci in bacterial pathogens.

Bacterial Typing Techniques↗

Staphylococcal chromosome cassette evolution in Staphylococcus aureus inferred from ccr gene complex sequence typing analysis.

Staphylococcal chromosome cassette mec (SCCmec) elements within major lineages of healthcare- and community-associated methicillin-resistant Staphylococcus aureus (MRSA) clones were characterised using intra-SCCmec multilocus sequencing. A strong correlation was observed between sequence- and PCR-based typing methods (p <0.001). However, phylogenetic analysis of the SCCmec locus using concatenated sequences evidenced few recombination events. Sequence type (ST)-SCCmec1 was found in SCCmec elements types I and IV, suggesting the evolution of an SCCmecI element into an SCCmecIV element. This coincided with the spread of the clone harbouring this SCCmec element into the community. No correlation was observed between ST-SCCmec lineage and MRSA lineage, confirming multiple acquisitions of SCCmec by S. aureus. This was exemplified by the SCCmecIV ST-SCCmec10 element, which was detected in all of the clonal complexes examined, including healthcare- and community-associated MRSA. The acquisition of this SCCmec element was five- to ten-fold more common than that of others. Models of MRSA clone evolution suggest that this SCCmec was first found in the paediatric clone.

DNA Primers↗

Rapid and sensitive typing of forensic stains by PCR amplification of polymorphic simple repeat sequences in case work.

After failure of conventional typing and multilocus DNA fingerprinting methods to compare a minute vaginal swab stain with blood from a murder victim and a suspect, we used enzymatic DNA amplification (polymerase chain reaction, PCR) to discriminate the DNAs by typing of simple sequence lengths polymorphisms. A mixed dinucleotide locus in the HLA-DRB gene region and three novel tetranucleotide polymorphisms located autosomally as well as on the human Y chromosome were used to exclude a jailed subject from a case of murder.

Alleles↗

Genetic polymorphism of Aspergillus fumigatus in clinical samples from patients with invasive aspergillosis: investigation using multiple typing methods.

The genotypes of 52 strains of Aspergillus fumigatus isolated from 12 patients with invasive aspergillosis were investigated using three typing methods (random amplified polymorphic DNA, sequence-specific DNA polymorphism, and microsatellite polymorphism) combined with multilocus enzyme electrophoresis. Isolates were from patients hospitalized in three different geographic areas (Lyon, France; Grenoble, France; and Milan, Italy). In each case, the genetic polymorphism of several colonies (two to five) within the first respiratory clinical sample was studied. For the 52 isolates tested, random amplified polymorphic DNA identified 8 different genotypes, sequence-specific DNA polymorphism identified 9 different types, and microsatellite polymorphism identified 14 types. A combination of these results with multilocus enzyme electrophoresis study identified 25 different types within the sample studied. We identified 3 patients (of the 12 studied) who carried a single genotype; 6 patients were infected by two genotypes, 1 patient had four genotypes, while the last patient had five. A combination of typing methods provided better discrimination than the use of a single method. Typing methods revealed a population structure within each geographical site, suggesting that the epidemiology of A. fumigatus should be considered separately for each of these geographic areas. This study demonstrates the usefulness of combining several typing methods in reaching an understanding of the epidemiology of A. fumigatus and clarifies whether it is sufficient to type one isolate from each specimen to determine the strain involved in invasive aspergillosis.

Aspergillosis↗

Yersinia aleksiciae sp. nov.

Yersinia kristensenii consists of phenotypically heterogeneous strains. This is reflected by the existence of strains with various multilocus enzyme electrophoresis and 16S rRNA gene sequence types. Strains originally phenotyped as members of Y. kristensenii were studied using 16S rRNA gene sequencing, DNA-DNA hybridization, determination of the DNA base composition and various phenotypic tests. The results were compared to those of Yersinia type strains. Based on levels of DNA-DNA relatedness, a specific 16S rRNA gene sequence type and the presence of lysine decarboxylase activity, a novel species, Yersinia aleksiciae sp. nov., is proposed. The type strain is Y159(T) (=WA758(T)=DSM 14987(T)=LMG 22254(T)).

Animals↗

A new highly discriminatory typing scheme for Treponema pallidum reveals similar levels of genetic variability across lineages.

UNLABELLED: The global resurgence of treponematoses, particularly syphilis, poses a growing public health challenge. Despite advances in sequencing technologies, obtaining complete Treponema pallidum genome sequences for epidemiological studies remains challenging due to clinical sampling and methodological constraints. There is, therefore, a need for rapid, cost-effective, and accessible typing methods. Based on the analysis of 121 T. pallidum genomes spanning all three subspecies (TPA, TPE, and TEN) from diverse regions, we selected seven highly variable genes (tp0136, tp0326, tp0548, tp0705, tp0858, tp0865, and tp1031) to form a new typing system, combined with analysis of macrolide resistance mutations in the 23S rRNA gene. The scheme was validated on 542 global T. pallidum samples, using either Sanger reads or whole genome sequence data, obtaining 82 sequence types (STs) among the 415 fully typed samples. Macrolide resistance mutations were frequently detected, highlighting the need for ongoing epidemiological surveillance. Phylogenetic analyses based on concatenated multilocus typing (MLST) loci recovered the expected subspecies and lineage structure. Consistently, almost all sequence types formed monophyletic groups, indicating strong concordance between MLST-based classification and whole-genome phylogenies. In addition, population genetic analyses revealed comparable levels of within-lineage diversity across subspecies and lineages, despite pronounced differences in geographic distribution, and identified distinct regional genetic clusters consistent with localized transmission dynamics. Importantly, the scheme employs a single-step PCR for all seven targets, facilitating implementation in standard laboratories and is publicly accessible through PubMLST. Overall, our novel MLST scheme offers a rapid, cost-effective tool to advance molecular epidemiology of T. pallidum, facilitate transmission and resistance tracking, and support global surveillance to strengthen public health interventions for syphilis and endemic treponematoses control. IMPORTANCE: We have developed a new multilocus typing (MLST) scheme useful for all Treponema pallidum lineages after the analysis of 121 complete genome sequences of this species. The new scheme can be used directly with uncultured clinical samples, thus providing an excellent contribution to the molecular surveillance of syphilis and other treponematoses. The application of this MLST scheme to over 500 samples from all lineages and main geographical regions has revealed similar levels of genetic variation within them. Furthermore, the analyses show a complex pattern of spread, with global and local contributions to the observed distribution of genetic variation in the syphilis-producing sublineages. The new scheme represents a significant improvement over previous proposals and also reveals unsuspected levels of variability in T. pallidum lineages.

Treponema pallidum↗

A decade of community MRSA in New Zealand.

In 1992, isolates with a distinctive phage pattern were identified amongst the 186 MRSA recovered in New Zealand. These unusual isolates were recovered in the Auckland region from individuals who came from or had visited Western Samoa, and were called Western Samoan phage pattern (WSPP) MRSA. They were almost exclusively community based and were mainly responsible for the alarming 15-fold increase in MRSA seen in New Zealand over the next 6 years. Since 2000, the number of infections attributable to WSPP MRSA appears to be declining. WSPP isolates are clonal, possess a unique type IV SCC mec element, and a distinctive multilocus sequence allelic profile (ST30). WSPP isolates are invariably not multiresistant with methicillin MICs generally <or=32 microg/ml. Virulence of the WSPP clone appears to be related to its adhesive and consistent toxin- (e.g. Panton-Valentine leukocidin, alpha- and gamma-haemolysins) producing capabilities. Isolates are most frequently associated with cutaneous lesions in younger age groups. Since 1998, MRSA isolates belonging to the UK-derived EMRSA-15 strain (also type IV SCC mec) have continued to increase in New Zealand, and together with WSPP, these strains now dominate MRSA isolations in New Zealand.

Community-Acquired Infections↗

Rat phenol-preferring sulfotransferase genes (Stp and Stp2): localization to mouse chromosomes 7 and 17.

The phenol-preferring sulfotransferases aryl sulfotransferase IV and N-hydroxyarylamine sulfotransferase catalyze sulfate conjugation of N-hydroxy-2-acetylaminofluorene, a metabolite capable of causing hepatocarcinogenesis in rats. We utilized published cDNA sequences of these sulfotransferases to type the progeny of two multilocus crosses and determined that the genes, aryl sulfotransferase (Stp) and N-hydroxyarylamine sulfotransferase (Stp2), map to positions on mouse chromosomes 7 and 17.

Animals↗

[DNA fingerprint method of the cultured cells].

DNA fingerprint method is very available not only to examine the cross-contamination of cultured cell lines and their origins and but to know the somatic mutation frequency in the cultured cells by exposure of radiation and chemicals. The method for DNA fingerprint was grouped in the following four types in term of probe; multilocus minisatellite probe, simple sequence repeat probe, locus specific minisatellite probe and repeat unit sequence variation in minisatellites.

Cell Line↗

Simple repeat sequences on the human Y chromosome are equally polymorphic as their autosomal counterparts.

The human genome contains a large number of interspersed simple repeat sequences that are variable in length and can therefore serve as highly informative, polymorphic markers. Typing procedures include conventional multilocus and single locus probing, and polymerase chain reaction aided analysis. We have identified simple sequences in a cosmid clone stemming from the human Y chromosome and consisting of (gata)n repeats. We have compared these with two equivalent simple repeat loci from chromosome 12. After amplifying the tandemly repeated motifs, we detected between four and eight different alleles at each of the three loci. Codominant inheritance of the alleles was established in family studies and the informativity of the simple repeat loci was determined by typing unrelated individuals. The polymorphisms are suitable for application in linkage studies, practical forensic case work, deficiency cases in paternity determination, and for studying ethnological questions. The mutational mechanisms that bring about changes in simple repeats located both on the autosomes and on the sex chromosomes, are discussed.

Base Sequence↗

Extensive polymorphism in Cryptosporidium parvum identified by multilocus microsatellite analysis.

Restriction fragment length polymorphism and DNA sequence analysis discern two main types of Cryptosporidium parvum. We present a survey of length polymorphism at several microsatellite loci for type 1 and type 2 isolates. A total of 14 microsatellite loci were identified from C. parvum DNA sequences deposited in public databases. All repeats were mono-, di-, and trinucleotide repeats of A, AT, and AAT, reflecting the high AT content of the C. parvum genome. Several of these loci showed significant length polymorphism, with as many as seven alleles identified for a single locus. Differences between alleles ranged from 1 to 27 bp. Karyotype analysis using probes flanking three microsatellites localized each marker to an individual chromosomal band, suggesting that these markers are single copy. In a sample of 19 isolates for which at least three microsatellites were typed, a majority of isolates displayed a unique multilocus fingerprint. Microsatellite analysis of isolates passaged between different host species identified genotypic changes consistent with changes in parasite populations.

Animals↗

Population structure of Salmonella investigated by amplified fragment length polymorphism.

AIMS: This study was undertaken to investigate the usefulness of amplified fragment length polymorphism (AFLP) in determining the population structure of Salmonella. METHODS AND RESULTS: A total of 89 strains were subjected to AFLP analysis using the enzymes BglII and BspDI, a combination that is novel in Salmonella. Both species S. bongori and S. enterica and all subsp. of S. enterica were represented with emphasis on S. enterica subsp. enterica using a local strain collection and strains from the Salmonella Reference Collection B (SARB). The amplified fragments were used in a band-based cluster analysis. The tree resulting from the subgroup analysis clearly separated all subgroups with high bootstrap values with the species S. bongori being the most distantly related of the subgroups. The tree resulting from the analysis of the SARB collection showed that some serotypes are very clonal whereas others are highly divergent. CONCLUSIONS: AFLP clearly clustered strains representing the subgroups of Salmonella together with high bootstrap values and the serotypes of subspecies enterica were divided into polyphyletic or monophyletic types corresponding well with multilocus enzyme electrophoresis (MLEE) and sequence-based studies of the population structure in Salmonella. SIGNIFICANCE AND IMPACT OF THE STUDY: AFLP with the enzyme combination BglII and BspDI allows discrimination of individual strains and provides evidence for the usefulness of AFLP in studies of population structure in Salmonella.

Base Sequence↗