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PubMed2005

Rapid genotypic detection of Bacillus anthracis and the Bacillus cereus group by multiplex real-time PCR melting curve analysis.

Bacillus anthracis has four plasmid possible virulence genotypes: pXO1+/pXO2+, pXO1+/pXO2-, pXO1-/pXO2+ or pXO1-/pXO2-. Due to the lack of a specific chromosomal marker for B. anthracis, differentiation of the pXO1-/pXO2- form of B. anthracis from closely related Bacillus cereus group species is difficult. In this study, we evaluate the ability of sspE, pXO1 and pXO2 primers to discriminate individual B. anthracis and the B. cereus group genotypes using multiplex real-time PCR and melting curve analysis. Optimal conditions for successful multiplex assays have been established. Purified DNAs from 38 bacterial strains including 11 strains of B. anthracis and 18 B. cereus group strains were analyzed. Nine of the B. cereus group near-neighbor strains were shown by multilocus sequence typing to be phylogenetically proximate to the B. anthracis clade. We have demonstrated that the four plasmid genotypes of B. anthracis and B. cereus group near-neighbors were differentially and simultaneously discriminated by this assay.

Bacillus anthracis↗

PubMed2004

Displaying the relatedness among isolates of bacterial species -- the eBURST approach.

Determining the most appropriate way to represent the relationships between bacterial isolates is complicated by the differing rates of recombination within species. In many cases, a bifurcating tree can be positively misleading. The recently described program eBURST can be used with multilocus data to define groups or clonal complexes of related isolates derived from a common ancestor, the patterns of descent linking them together, and the ancestral genotype. eBURST has recently been extensively updated to include additional tools for exploring the relationships between isolates. We discuss the advantages of this approach and describe its use to explore patterns of descent within clonal complexes identified using multilocus sequence typing.

Algorithms↗

PubMed2006

Nosocomial infections by Staphylococcus epidermidis: how a commensal bacterium turns into a pathogen.

Staphylococcus epidermidis is a commensal bacterium of the human skin. However, S. epidermidis and other coagulase-negative staphylococci (CNS) emerge also as common nosocomial pathogens infecting immunocompromized patients carrying medical devices. Antibiotic resistance and the ability of many nosocomial S. epidermidis isolates to form biofilms on inert surfaces make these infections hard to treat. Epidemiological analyses using multilocus sequence typing (MLST) and genetic studies suggest that S. epidermidis isolates in the hospital environment differ from those obtained outside of medical facilities with respect to biofilm formation, antibiotic resistance, and the presence of mobile DNA elements. Since S. epidermidis isolates exhibit high genome flexibility, they are now regarded as reservoirs for the evolution and spread of resistance traits within nosocomial bacterial communities.

Biofilms↗

PubMed2004

Population structure of pathogenic bacteria revisited.

This minireview summarizes the historical development of bacterial population genetic concepts since the early 1980s. Initially multilocus enzyme electrophoresis was used to determine population structures but this technique is poorly portable between laboratories and was replaced in 1998 by multilocus sequence typing. Diverse population structures exist in different bacterial species. Two distinctive structures are described in greater detail. "Young" organisms, such as Yersinia pestis, have evolved or undergone a severe bottleneck in recent millennia and have not yet accumulated much sequence diversity. "genoclouds" in subgroup III Neisseria meningitidis arise because of the accumulation of diversity due to herd immunity, which is then purified during subsequent epidemic spread.

Bacteria↗

PubMed2025

Selecting candidate Neisseria gonorrhoeae strains for oropharyngeal gonorrhoea human challenge: a genomics-based analysis of clinical isolates.

BACKGROUND: Neisseria gonorrhoeae is a human pathogen of major public health importance due to its increasing global prevalence and antimicrobial resistance (AMR). Evidence suggests that oropharyngeal infection plays a key role in N gonorrhoeae transmission and AMR; however, our understanding of oropharyngeal gonorrhoea pathogenesis is poor. A controlled human infection model (CHIM) for oropharyngeal gonorrhoea will improve understanding of infection and accelerate urgently needed novel gonorrhoea prevention and therapeutic strategies. As the first step in the development of this CHIM, we describe a systematic approach to CHIM strain selection that leverages genomics and clinical data. METHODS: In this genomics-based analysis, we applied a systematic N gonorrhoeae challenge strain selection strategy incorporating genomic and clinical data to a primary dataset of clinical isolates of N gonorrhoeae collected from adult patients in Victoria, Australia, between Jan 1 and Dec 31, 2017, and July 1, 2019, and June 30, 2021. This selection strategy used clinical, phenotypic, and genomic characteristics to define a set of eight criteria that aimed to ensure the contemporary global clinical relevance of the candidate strains; select strains that would be applicable for the assessment of current and future gonorrhoea vaccines; and maximise participant safety by reducing the risk of disseminated gonococcal infection and clinically significant AMR. We applied these criteria to our primary dataset to generate a panel of potential challenge strains. From this final dataset of potential challenge strains, we predetermined that we would select up to ten isolates to proceed to the next stage of detailed phenotypic characterisation for final N gonorrhoeae CHIM strain selection. FINDINGS: 5881 isolates comprised the primary dataset. After application of the selection criteria, most of the isolates (5795 [98·6%] of 5881) were excluded, mostly due to having clinically significant AMR and poor contemporary global clinical relevance. The remaining 86 N gonorrhoeae challenge strain candidates comprised five multilocus sequence types and six N gonorrhoeae multiantigen sequence types, many of which were represented by a single isolate. Of these 86 strains, five isolates were selected to maximise coverage of the phylogenetically distinct groups within the 86 candidate challenge strains and ensure representation of strains collected from various anatomical sites. INTERPRETATION: We transparently describe a novel, systematic, and rational genomics-based strategy for oropharyngeal gonorrhoea CHIM strain selection that improves the efficiency and transparency of CHIM strain selection and enables identification of contemporary and clinically relevant potential challenge strains. A final N gonorrhoeae challenge strain will be selected from the subset of five shortlisted candidates after detailed phenotypic assessment. FUNDING: Medical Research Future Fund, Australian National Health and Medical Research Council and Australian Government Research Training Program.

Humans↗

PubMed2025

Universal versus targeted chlorhexidine and mupirocin decolonisation and clinical and molecular epidemiology of Staphylococcus epidermidis bloodstream infections in patients in intensive care in Scotland, UK: a controlled time-series and longitudinal genotypic study.

BACKGROUND: There are concerns that biocide skin and mucous membrane decolonisation, which is widely used to prevent health-care-associated infections in intensive care units (ICUs), might select for multidrug-resistant pathogens. We aimed to evaluate the effects of de-escalating from universal to targeted skin and nasal decolonisation on Staphylococcus epidermidis bloodstream infections (SE-BSI). METHODS: We did a retrospective, before-after-control-impact time-series analysis and longitudinal genotypic study in two ICUs with divergent decolonisation practice in tertiary care hospitals of adjacent health boards in Scotland, UK. Participants were aged at least 16 years and admitted between July 1, 2009, and Feb 28, 2022. There were no exclusion criteria for the study. In ICU one (intervention site) universal decolonisation in all admissions was de-escalated to targeted decolonisation of meticillin-resistant Staphylococcus aureus (MRSA) carriers on Feb 1, 2019, while in ICU two (control site) targeted decolonisation was applied throughout. We collected bloodstream infection data from all causes, including clinically significant SE-BSI. Antimicrobial susceptibility testing was used to define meticillin-resistant S epidermidis (MRSE) and chlorhexidine susceptibility. We used multilocus sequence typing to identify sequence types from archived SE-BSI isolates. Whole-genome sequencing was applied to a sample from ICU one. The primary outcomes were incidence densities of all bloodstream infections, SE-BSI, and meticillin-resistant S epidermidis bloodstream infections (MRSE-BSI), and the percentage probability that SE-BSI were MRSE-BSI. The effects of de-escalation on primary outcomes were estimated by differences between the intervention and control sites, before and after de-escalation, using a before-after-control-impact time-series design. Secondary outcomes included the proportion of multidrug resistant sequence types, carriage of mobile genetic elements and genes for multidrug resistance and biofilm production. FINDINGS: Between July 1, 2009, and Feb 28, 2022, S epidermidis was identified in 334 (45%) of 735 bloodstream infections in ICU one, of which 197 occurred before the de-escalation intervention in Feb 1, 2019, and S epidermidis was identified in 167 (60%) of 278 bloodstream infections in ICU two. There was no increase in all bloodstream infection incidence coinciding with de-escalation in ICU one, whereas MRSE-BSI incidence declined significantly from 10·4 cases per 1000 occupied bed days (OBDs; 95% credible interval [CrI] 7·2-15·4) to 4·3 cases per 1000 OBDs (2·5-6·7), as did the percentage probability of MRSE (from 89·2%, 95% CrI 77·8-96·5 to 56·7%, 34·3-77·5%). No significant changes in the primary outcomes were seen in ICU two. MRSE-BSI incidence density was positively associated with chlorhexidine use, but not mupirocin use. De-escalation was associated with a reduced proportion of SE-BSI due to multidrug-resistant sequence types and reduced carriage of mobile genetic elements and genes for multidrug resistance and biofilm production, as observed by multi-locus sequence typing and whole genome sequencing. INTERPRETATION: In ICU settings with low MRSA incidence, the benefits of universal decolonisation should be balanced against the risks of selecting MRSE sequence types adapted for invasive and device-associated infection. FUNDING: National Health Service Grampian Charity.

Humans↗

PubMed2004

Analyses of clonality and the evolution of bacterial pathogens.

The existence of bacterial clones was evident in early phenotypic studies that recognised high levels of similarity in geographically and temporally separated isolates. Multilocus sequence typing (MLST) has become the most common method for genetically characterizing clones of several bacterial pathogens, allowing the tracking of hypervirulent/antibiotic-resistant lineages. MLST has also been used to examine the way that bacterial populations, and in particular, bacterial clones evolve. Visualisation of MLST datasets has required the development of novel tools, such as 'eBURST', a key program in constructing evolutionary models that detail how methicillin resistant Staphylococcus aureus (MRSA) and other clones emerge and spread.

Algorithms↗

PubMed2006

Identification of high-risk enterococcal clonal complexes: global dispersion and antibiotic resistance.

Vancomycin-resistant Enterococcus faecium spread dramatically in hospital settings in the USA in the 1990s and reached endemicity at the turn of the century. Similarly, rising prevalence rates are currently observed in several European countries, with prevalence rates of greater than 10% reported in seven of these. On the basis of multilocus sequence typing (MLST), the population structure of E. faecium was elucidated and the existence of a distinct high-risk enterococcal clonal complex, designated clonal complex-17 (CC17), which is associated with the majority of hospital outbreaks and clinical infections in five continents, was revealed. This complex is correlated with ampicillin and quinolone resistance and with the presence of a putative pathogenicity island. Preliminary MLST data suggest that similar hospital-adapted complexes might also exist in E. faecalis.

Ampicillin Resistance↗

PubMed2006

Genomic diversity and evolution within the species Streptococcus agalactiae.

Streptococcus agalactiae is a leading cause of invasive infections in neonates, and responsible for bovine mastitis. It is also a commensal bacterium adapted to asymptomatic colonization of the mammalian gut and of the genitourinary tract. Here, we report the analysis of a collection of 75 strains of human and animal origin by using serotyping, multilocus sequence typing, whole genome DNA-array hybridizations and sequence comparison of putatively virulence-associated loci. Although the most variable parts of the genome are the previously predicted genomic islands, significant genetic variations were present in the genome backbone. Evolution within genes encoding surface and secreted proteins and those involved in the biosynthesis of different capsular types is mainly due to recombination events leading to the replacement of a locus of several genes or to the allelic exchange of the internal part of a gene. These two processes, which led to a broad diversity of surface protein patterns, are probably involved in the diversity of interactions with the host and its immune system. According to gene content comparisons and phylogeny, recent gene replacements by horizontal gene transfer may occur but are rare events. Although specific gene patterns, with respect to the origin of the strains and the epidemiological characteristics, were not identified, we show that the recently described hypervirulent ST-17 lineage is a homogeneous group. The study highlights for the first time that this lineage contains a specific and conserved set of surface proteins, probably accounting for its high capacity to cause infections in newborns.

Adult↗

PubMed2006

Rapid detection of the "highly virulent" group B Streptococcus ST-17 clone.

Group B streptococcus (GBS) is a leading cause of neonatal morbidity and mortality. Multilocus sequence typing (MLST) revealed that the sequence type ST-17 defines a "highly virulent" serotype III clone strongly associated with neonatal invasive infections. Our aim was to identify a target sequence enabling rapid, simple, and specific detection of this clone by a real-time PCR assay. Conventional methods for DNA manipulation and gene analyses were used to characterize the gbs2018 gene variant specific for ST-17 clone and to design ST-17- and GBS-specific primers. Conventional and real-time PCR assays were developed to detect GBS and ST-17 clones in bacterial cultures and directly on clinical samples. One hundred and fifty-six French GBS strains from various geographical areas in France isolated between 1990 and 2005 were screened by PCR with ST-17-specific primers. Forty strains were positive, and all were validated by MLST as ST-17. A representative sampling of 49 ST-17-PCR-negative strains was confirmed by MLST as non-ST-17. Real-time PCR was further used to directly test 85 vaginal samples. Among these, 13 were GBS-positive, and one was identified as ST-17. The association between strain invasiveness and ST-17 lineage in neonates with late onset disease was highly significant: 78% (P<0.0001) of strains isolated were ST-17. In conclusion, an ST-17-specific gbs2018 allele was identified and used to develop a sensitive and specific rapid-screening molecular assay for identifying ST-17 "highly virulent" GBS. Using this technique, accurate identification of women and neonates colonized by ST-17 can be readily achieved within less than 2 h.

Adult↗

PubMed2026

Prevalence, genomic characterization, and biofilm-forming capacity of extended-spectrum β-lactamase-producing Escherichia coli from faecal samples of broiler chickens in Jinan City, China.

The emergence of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli in food animals threatens both veterinary and human medicine by compromising critically important antimicrobials. This study characterized the prevalence, antimicrobial resistance profiles, molecular epidemiology, and virulence attributes of ESBL-producing E. coli isolated from broiler farms in Jinan City, China. From 600 faecal samples, 537 E. coli isolates were recovered (89.5 % isolation rate), with 71 (13.2 %) identified as ESBL producers. Antimicrobial susceptibility testing revealed markedly more severe resistance among ESBL-producing isolates, with complete ampicillin resistance (100 %) and high-level resistance to sulfamethoxazole/trimethoprim (94.37 %), streptomycin (87.32 %), chloramphenicol (78.87 %), and tetracycline (70.42 %). Resistance to extended-spectrum cephalosporins cefuroxime and ceftriaxone reached 43.66 % and 36.62 %, respectively, while amoxicillin/clavulanic acid susceptibility declined to 61.97 %. Whole-genome sequencing identified blaCTX-M-55 (29.58 %) as the predominant ESBL genotype, followed by blaCTX-M-64 (23.94 %), blaCTX-M-15 (19.71 %), blaCTX-M-14 (12.68 %), and blaCTX-M-65 (9.86 %). The plasmid-mediated colistin resistance gene mcr-1 was detected in 25.35 % of ESBL-producing isolates, indicating a substantial reservoir of last-resort antibiotic resistance determinants, though physical linkage between mcr-1 and ESBL-encoding genes remains undetermined due to short-read sequencing limitations. Multilocus sequence typing revealed ST117 (18.3 %) as the predominant sequence type, associated mainly with serogroup O78 (25.4 %). Virulence gene profiling demonstrated high prevalence of the serum survival gene iss (85.9 %), commonly associated with avian pathogenic E. coli, though avian pathogenicity was not experimentally confirmed. Notably, 90.14 % of ESBL-producing isolates demonstrated biofilm-forming capacity, with 16.90 % classified as strong biofilm producers forming mature, mushroom-shaped microcolonies. These findings demonstrate that broiler chickens in the sampled Jinan farms constitute a significant reservoir of multidrug-resistant, biofilm-forming ESBL-producing E. coli harboring clinically relevant genotypes, including mcr-1. The predominance of ST117, reported in both poultry and human clinical settings, along with extensive co-resistance profiles, underscores the value of integrated surveillance and antimicrobial stewardship in poultry production, though direct zoonotic transmission evidence requires further comparative genomic and epidemiological investigation.

Antimicrobial resistance↗

PubMed2004

[Clostridium difficile, nosocomial enteropathogen: phylogeny and virulence].

Clostridium difficile is recognized as a potentially nosocomial enteric pathogen. It induces diarrhea or pseudomembranous colitis in patients whose digestive flora has been altered by antibiotic treatment and thus allows the colonization with a strain producing toxin A (enterotoxin) and toxin B (cytotoxin) (A+B+, sometimes A-B+ strains). We studied the phylogeny of C. difficile by developing MultiLocus Sequence Typing (MLST) analysis, which reports allelic polymorphism of housekeeping genes through DNA sequencing. C. difficile exhibits genomic stability, with mutational clonal evolution and individualization of phylogenetic lineages. These lineages are not correlated with human or animal hosts. Strains involved in pseudomembranous colitis or in diarrhea do not define distinct lineages, and bi-toxinogenic strains do not segregate from non toxinogenic strains. Conversely, A-B+ strains define a unique clone, highly divergent from the population studied. Allelic sequence data may be available from an centralized internet site, allowing phylogenetic and macro-epidemiologic analyses.

Biological Evolution↗

PubMedDate not supplied

Estimating the relative contributions of mutation and recombination to clonal diversification: a comparison between Neisseria meningitidis and Streptococcus pneumoniae.

Both Neisseria meningitidis and Streptococcus pneumoniae are naturally transformable species and are known to be freely recombining in the wild. Large multilocus sequence typing (MLST) datasets have been generated for these species. Here we outline an approach which exploits these data sets in order to quantify the extent of recombination, thus enabling meaningful comparisons between the two species. Two parameters are estimated; the rate at which recombination changes alleles, compared to point mutation, and the rate at which recombination changes individual nucleotide sites, compared to point mutation. Estimates for the former parameter are 4:1 in the meningococcus (i.e. alleles are changed four-fold more frequently by recombination than by mutation), and 10:1 in the pneumococcus. However, estimates for the latter parameter are at least 80:1 in the meningococcus (i.e. an individual nucleotide site is at least 80-fold more likely to change by recombination than by mutation) and 50:1 in the pneumococcus. These data imply that recombination events, compared to mutational events, may be more common in the pneumococcus than in the meningococcus. However, because it is a more diverse species, each recombinational exchange in the meningococcus results in more nucleotide changes on average.

Genetic Variation↗

PubMed2001

The relative contributions of recombination and point mutation to the diversification of bacterial clones.

Low levels of recombination in bacterial species have often been inferred from the presence of linkage disequilibrium between the alleles at different loci in the population. However, significant linkage disequilibrium is inevitable in organisms that divide by binary fission, and recombinational replacements must be very frequent, compared to point mutation, to dissipate disequilibrium. Recent studies using data from multilocus sequence typing indicate that, in many species, recombinational replacements contribute more greatly to clonal diversification than do point mutations and, in some species, recombination has been sufficient to eliminate any phylogenetic signal from gene trees. Recent efforts to improve understanding of the extent and impact of homologous recombination in the diversification of bacterial clones are discussed.

Bacteria↗

PubMed2001

Characteristics of a new epidemic MRSA in Germany ancestral to United Kingdom EMRSA 15.

In 1996 a new epidemic MRSA emerged in three hospitals North of Berlin. This strain, Barnim epidemic MRSA, was isolated in 15 hospitals in Northern Germany in 1997 and 29 hospitals throughout Germany in 1998. Isolates of this clone are non-typeable by phages, its resistance phenotype is PEN, OXA, ERY, CLI, CIP (genotype: mecA, ermC, mutations in grlA and gyrA). The Sma I macrorestriction pattern corresponds to particular phage group II strains which is confirmed by the 16S-23S rRNA gene spacer pattern. Isolates of this clone differ by less than three Sma I macrorestriction fragments from isolates of the EMRSA15 clone from the United Kingdom, the most common epidemic MRSA isolates in the United Kingdom in recent years. Both epidemic strains produce enterotoxin C and possess the sec determinant for this toxin, the configuration of the mec regulon is mecI-, mecRB+, mecRC+. Both share the same Alu I pattern of PCR amplimers of the 3' end region of the coagulase gene. EMRSA 15 and Barnim EMRSA share a common multilocus sequence type indicating a recent, shared evolutionary origin.

Bacterial Typing Techniques↗

PubMed2001

Genetics and genomics in infectious disease susceptibility.

The past decade has witnessed a rapid transition from the first positional cloning of an infectious disease susceptibility gene (Slc11a1, also called Nramp1) in the mouse to genome-wide scans in human multicase families and the identification of potential disease-causing genes by simple inspection of the public human genome databases. Pathogen genome projects have facilitated multilocus sequence typing of pathogen isolates and studies of ecological fitness and virulence patterns in disease-causing isolates. Comparative sequence analysis of pathogen strains and functional genomics studies are now underway, hopefully providing new insight into infectious disease susceptibility.

Cloning, Molecular↗

PubMed2001

The population structure of Neisseria meningitidis serogroup A fits the predictions for clonality.

The population structure of Neisseria meningitidis is supposedly epidemic according to. The model predicts that linkage disequilibrium in N. meningitidis populations is only temporary and arises due to the outgrowth of highly successful clonal genotypes from an essentially sexual population. These clones should disappear after a few years because of frequent recombination. In contrast, multilocus enzyme electrophoresis (MLEE) data had previously been interpreted as showing that serogroup A meningococci are truly clonal and possess only limited genetic variability (Wang et al., 1992). The two interpretations are contradictory. In order to elucidate the true population structure of serogroup A meningococci, we analyzed data for a representative group of 84 serogroup A isolates obtained by MLEE, random amplified polymorphic DNA (RAPD) and multilocus sequence typing (MLST). Analysis of linkage disequilibrium and bootstrap analyses of cluster analysis showed a strongly structured population with highly significant linkage disequilibrium. This was not due to the overrepresentation of certain genotypes, in contrast to the expectations for an epidemic population. The analyses identify two main clades, within each of which linkage disequilibrium was also highly significant, thus, excluding a cryptic speciation model. These observations support a population structure based on clonal evolution, in which clones are much more stable than expected for epidemic clonality. We propose that serogroup A meningococci may possess a different population structure from other serogroups of Neisseria meningitidis.

Clone Cells↗

PubMed2003

Coagulase gemne variants associated with distinct populations of Staphylococcus aureus.

An identifying characteristic of Staphylococcus aureus is the production of staphylocoagulase (coagulase). The aim of this study was to determine the clonal distribution of coagulase gene (coa) variants within populations of S. aureus defined by multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and protein A variation. The N-terminal region of the coa gene from 43 methicillin-susceptible (MSSA) and 252 methicillin-resistant (MRSA) S. aureus human isolates and 9 animal S. aureus isolates was amplified and digested with HinfI. Twelve types were identified amongst the MSSA isolates and the majority (93%) of MRSA isolates were assigned to 5 of the 12 types. MLST and PFGE analysis identified epidemic populations of MRSA and each epidemic population was characterized by a different coagulase type. Nine of the 12 MLST-defined clonal complex ancestral genotypes recently described each carried a different coagulase type suggesting that coagulase evolution and the evolution of the clonal complexes are intimately related.

Amino Acid Sequence↗