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PubMed2015

Impermanence of bacterial clones.

Bacteria reproduce asexually and pass on a single genome copied from the parent, a reproductive mode that assures the clonal descent of progeny; however, a truly clonal bacterial species is extremely rare. The signal of clonality can be interrupted by gene uptake and exchange, initiating homologous recombination that results in the unique sequence of one clone being incorporated into another. Because recombination occurs sporadically and on local scales, these events are often difficult to recognize, even when considering large samples of completely sequenced genomes. Moreover, several processes can produce the appearance of clonality in populations that undergo frequent recombination. The rates and consequences of recombination have been studied in Escherichia coli for over 40 y, and, during this time, there have been several shifting views of its clonal status, population structure, and rates of gene exchange. We reexamine the studies and retrace the evolution of the methods that have assessed the extent of DNA flux, largely focusing on its impact on the E. coli genome.

Clone Cells↗

PubMed2025

Molecular insights into carbapenemase-producing Enterobacterales from Senegal.

BACKGROUND: Carbapenemase-producing Enterobacterales (CPE) are an urgent global health threat, especially in resource-limited countries. Here we determined the prevalence and the molecular characteristics of CPE isolated from infections in Dakar, Senegal. METHOD: From January 2019 to December 2020, Enterobacterales with reduced susceptibility to ertapenem (diameter < 25 mm) from infections were collected at the Pasteur Institute of Dakar. Carbapenemases were detected using biochemical and immunochromatographical assays. WGS was used to determine resistome, MLST, plasmids, virulence genes and genetic relatedness. RESULTS: Of the 1045 Enterobacterales collected during the study period, 86 had a diameter around ertapenem of <25 mm (8%) and 19 were confirmed as CPE (2%). These included Escherichia coli (n = 6) [ST410 (n = 3), ST405 (n = 2) and ST2083], Enterobacter spp. (n = 6) [ST231 (n = 3), ST245, ST760 and ST960] and Klebsiella spp. (n = 5) (ST22, ST25, ST231, ST1535, ST4843), Citrobacter freundii ST22 (n = 1) and Citrobacter koseri with unknown ST (n = 1). blaOXA-48 (n = 7; 35%), blaOXA-181 (n = 7; 35%) and blaNDM-5 (n = 6; 30%) genes were identified. C. freundii ST22 harboured blaNDM-5, blaOXA-48 and blaCTX-M-15 genes. Some E. coli isolates belonging to the high-risk clone ST410 were closely related (<20 SNPs) to isolates recovered in France from patients returning from Senegal, suggesting transnational spread. In addition, 5/6 carbapenemase-producing E. coli isolates possessed a four amino acid insertion in PBP3, conferring reduced susceptibility to aztreonam/avibactam and cefiderocol. CONCLUSIONS: This study highlights the spread of NDM-5 and OXA-181 in Senegal, and reports the first co-occurrence of NDM-5 and OXA-48 in sub-Saharan Africa. The spread of CPE, especially in high-risk clones, underscores the urgent need for continued surveillance and targeted interventions.

Senegal↗

PubMed2026

Genomic Characterization of Aeromonas dhakensis Isolated From a Fatal Dolphin Case.

Aeromonas dhakensis has emerged as a significant pathogen affecting both aquatic animals and humans; however, genomic data for isolates from marine mammals remain scarce. In this study, we characterised the genome of A. dhakensis strain KDL-001, isolated from a fatal dolphin case, using whole-genome sequencing and comparative genomics. Taxonomic analyses, including MLST and average nucleotide identity (ANI), confirmed the isolate as A. dhakensis. Core-genome phylogeny further revealed that KDL-001 is closely related to strains derived from fish and aquatic environments. Notably, in silico screening of virulence-associated genes showed that the virulence-associated gene profile of the dolphin isolate was broadly comparable to those of other A. dhakensis strains, with no isolate-specific virulence-associated genes being identified within the limits of this analysis. These findings demonstrate that the dolphin-derived isolate is genomically comparable to previously described A. dhakensis strains and possesses conserved virulence-associated genes commonly found within the species.

Animals↗

PubMed2004

Phylogeny and strain typing of Escherichia coli, inferred from variation at mononucleotide repeat loci.

Multilocus sequencing of housekeeping genes has been used previously for bacterial strain typing and for inferring evolutionary relationships among strains of Escherichia coli. In this study, we used shorter intergenic sequences that contained simple sequence repeats (SSRs) of repeating mononucleotide motifs (mononucleotide repeats [MNRs]) to infer the phylogeny of pathogenic and commensal E. coli strains. Seven noncoding loci (four MNRs and three non-SSRs) were sequenced in 27 strains, including enterohemorrhagic (six isolates of O157:H7), enteropathogenic, enterotoxigenic, B, and K-12 strains. The four MNRs were also sequenced in 20 representative strains of the E. coli reference (ECOR) collection. Sequence polymorphism was significantly higher at the MNR loci, including the flanking sequences, indicating a higher mutation rate in the sequences flanking the MNR tracts. The four MNR loci were amplifiable by PCR in the standard ECOR A, B1, and D groups, but only one (yaiN) in the B2 group was amplified, which is consistent with previous studies that suggested that B2 is the most ancient group. High sequence compatibility was found between the four MNR loci, indicating that they are in the same clonal frame. The phylogenetic trees that were constructed from the sequence data were in good agreement with those of previous studies that used multilocus enzyme electrophoresis. The results demonstrate that MNR loci are useful for inferring phylogenetic relationships and provide much higher sequence variation than housekeeping genes. Therefore, the use of MNR loci for multilocus sequence typing should prove efficient for clinical diagnostics, epidemiology, and evolutionary study of bacteria.

Bacterial Typing Techniques↗

PubMed2000

[Multilocus sequencing--a new method of genotyping bacteria and first results of its use].

Comparative characterization (molecular typing) of isolates within a bacterial species is one of the major problems in microbiology and epidemiology. However, it is rather difficult to correlate data obtained in various laboratories, because traditional, including molecular, methods employed in typing pathogenic microorganisms cannot be standardized. In 1998, Maiden et al. proposed multilocus sequence typing (MLST); through which alleles of several housekeeping genes are directly assessed by nucleotide sequencing, each unique allele combination determining a sequence type of a strain. The advantages of this approach are that the culturing of pathogenic microorganisms is avoided, as their gene fragments are amplified directly from biological samples, and that the sequencing data are unambiguous, easy to standardize, and electronically portable. The latter makes it possible to generate an expandable global database for each species at an Internet site, in order to use it for the purposes of genotyping pathogenic bacteria (and other infectious agents). MLST protocols have been elaborated for Neisseria meningitidis, Streptococcus pneumoniae, and Helicobacter pylori; those for Streptococcus pyogenes, Staphylococcus aureus, and Haemophilus influenzae are now being developed. Basic principles and the first results of MLST have been reviewed, including data on the distribution and microevolution of N. meningitidis clones causing epidemic meningococcal infection, the relative recombination and mutation rates in the N. meningitidis genome, the identification of antibiotic-resistant S. pneumoniae clones causing severe generalized infection, the grouping of H. pylori isolates from various geographic regions, etc.

Bacteria↗

PubMed1994

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↗

PubMed1997

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↗

PubMed2004

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↗

PubMedDate not supplied

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↗

PubMed1992

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↗

PubMed2001

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↗

PubMed2026

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↗

PubMed1995

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↗

PubMed1993

[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↗

PubMed1992

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↗

PubMed2000

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↗

PubMed2004

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↗

PubMed2025

Understanding recurrence in Mycobacterium avium complex pulmonary disease: genotypic strategies to support clinical decision-making.

Pulmonary disease caused by Mycobacterium avium complex (MAC-PD) is a chronic, recurrent disease, and its high recurrence rate after treatment makes clinical management difficult. Distinguishing whether recurrence is due to persistence of existing strains or reinfection with new strains is essential for establishing treatment strategies, preventing overuse of antimicrobials, and establishing infection control measures. According to reports, 54%-74% of MAC-PD recurrence is due to reinfection, which may be mainly related to environmental reservoirs such as household water supply. In this review, we present various clinical scenarios in which MAC-PD recurrence may occur and examine genotyping techniques as a strategy to distinguish and respond to them. From traditional methods such as IS1245-based restriction fragment length polymorphism, pulsed-field gel electrophoresis, and hsp65 and rpoB gene sequencing to high-resolution analysis techniques such as multilocus sequence testing and whole-genome sequencing, the latest molecular typing methods are comprehensively summarized. Integrating these genotype data into clinical settings, standardizing single-nucleotide polymorphism-based interpretation thresholds, and promoting the establishment of a global MAC strain database will make a substantial contribution to more accurately distinguishing the recurrence mechanisms of MAC-PD and establishing personalized treatment strategies.IMPORTANCEThe global burden of nontuberculous mycobacterial pulmonary disease (PD) is increasing, with Mycobacterium avium (MAC)-PD being the most prevalent and clinically challenging form. Its low treatment success rates, high frequency of recurrence, and persistent environmental exposure complicate both diagnosis and management. A critical clinical issue is determining whether recurrence represents true relapse, due to persistence of the original strain, or reinfection with a new strain, as this guides treatment and prevents overtreatment. Genotypic strategies capable of resolving strain-level differences can improve diagnostic accuracy, prevent misclassification, and ultimately support more informed treatment decisions. Therefore, integrating genotyping data into clinical workflows, standardizing single-nucleotide polymorphism thresholds, and establishing a global MAC strain database will not only support personalized treatment but also enhance the broader public health response to this disease.

Humans↗