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PubMed2004

Staphylococcus aureus: superbug, super genome?

Staphylococcus aureus is a common cause of infection in both hospitals and the community, and it is becoming increasingly virulent and resistant to antibiotics. The recent sequencing of seven strains of S. aureus provides unprecedented information about its genome diversity. Subtle differences in core (stable) regions of the genome have been exploited by multi-locus sequence typing (MLST) to understand S. aureus population structure. Dramatic differences in the carriage and spread of accessory genes, including those involved in virulence and resistance, contribute to the emergence of new strains with healthcare implications. Understanding the differences between S. aureus genomes and the controls that govern these changes is helping to improve our knowledge of S. aureus pathogenicity and to predict the evolution of super-superbugs.

Drug Resistance, Bacterial↗

PubMed2006

Multilocus-sequence typing analysis reveals similar populations of Streptococcus uberis are responsible for bovine intramammary infections of short and long duration.

Multilocus-sequence typing (MLST) was used to analyse Streptococcus uberis isolates from a single herd associated with long duration (50-260 days) and rapidly cleared (less than 1 month) bovine intramammary infections to determine whether the bacterial type had any impact on the duration of infection. Most chronic infections (24 of 33) were due to continuous infection of the mammary quarter with the same sequence type, and infections were found to persist for many months. The remaining quarters were re-infected with a different sequence type within a single lactation. No particular sequence type or clonal complex (lineage) was associated with persisting infections, indicating that the outcome of intramammary infections with S. uberis is more likely to be dependent on host factors than on inter-strain differences. Analysis of these strains alongside others obtained from the same herd at a later date revealed the shift in the predominant genotypes with time.

Alleles↗

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↗

PubMed2003

Molecular epidemiology in the public health and hospital environments.

The increasing speed and ease of genomic sequencing coupled with available funding to sequence multiple, unrelated strains of the same species will lead inevitably to the identification of candidate genes that can be used as molecular typing tools (MLST, SLST, microarray approach). However, it is important to note that even the most sophisticated typing tool should never replace a full epidemiologic investigation in which all available information is taken into account. Nevertheless, the typing methods discussed in this article and those yet to be developed have significantly improved the quality of health care worldwide.

Bacterial Infections↗

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 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↗

PubMed2006

Antimicrobial susceptibility, serotype and genotype distribution of meningococci in Portugal, 2001-2002.

One hundred and eighteen Neisseria meningitidis isolates were recovered from patients with invasive meningococcal disease in Portugal, over one year. Our study was undertaken to evaluate antimicrobial susceptibility, serogroup, serotype and genotype of isolates. One quarter (24.6%) of the isolates showed moderate resistance to penicillin and 47.4% were resistant to sulphadiazine. The two most common serosubtypes were C:2b:P1.5,2 (31.3%) and B:4:P1.15 (3.4%). Half (53.6%) of the isolates with moderate resistance to penicillin were phenotype C:2b:P1.5,2 (n=14), C:2b:P1.2 (n=1) or C:2b:NST (n=1); Pulsed-field gel electrophoresis (PFGE) showed that all these isolates were genetically related. Multilocus sequence typing (MLST) analysis of representative clones from each PFGE pattern showed the predominance of the ST-8 complex/cluster A4 among N. meningitidis with moderate resistance to penicillin. This clonal complex has been principally found in Southern Europe. The apparent emergence and dissemination of the hypervirulent ST-8 complex/cluster A4 among serogroup C strains increases the need for a continued surveillance of antimicrobial susceptibility of meningococci and of genotypic markers in Portugal.

Anti-Infective Agents↗

PubMed2003

Molecular typing for fungi--a critical review of the possibilities and limitations of currently and future methods.

Invasive fungal infections represent an increasing problem in patients with inherited and acquired immunodeficiencies. Molecular biotyping techniques, such as DNA fingerprinting, are useful tools to increase our knowledge of the pathogenic organisms that cause them, and thus to improve their treatment and develop prevention strategies. In the present review, we evaluate and discuss the possibilities and limitations of the methods currently used for biotyping strains of fungal species. These include techniques based on restriction fragment length polymorphism (RFLP) with or without hybridization to probes (Southern), PCR-based techniques, electrophoretic karyotyping (EK), and multilocus enzyme electrophoresis (MLEE). Additionally, we discuss newer techniques that are being developed for the fingerprinting of fungal strains. Among them, we review conformation-based polymorphism scanning methods, such as single-strand conformation polymorphism analysis (SSCP) and heteroduplex mobility assays, sequencing strategies such as multilocus sequence typing (MLST) and DNA microarrays.

DNA Fingerprinting↗

PubMed2005

Streptococcal toxic shock syndrome by an iMLS resistant M type 77 Streptococcus pyogenes in the Netherlands.

An increasing number of group A streptococci (GAS) with constitutive or inducible resistance to macrolide-lincosamide-streptogramin B antibiotics (cMLS or iMLS phenotype) is observed in Europe, but MLS resistant GAS associated with streptococcal toxic shock syndrome (STSS) has not been reported. We describe a patient admitted with STSS caused by an iMLS resistant T28 M77 Streptococcus pyogenes carrying the ermA [subclass TR] gene. A 2-y retrospective analysis among 701 nationwide collected GAS strains revealed an incidence of 3.1% of this M type 77 GAS. Analysis of 17 available M77 strains (12 T28 and 5 T13) indicated that 2 (12%) were MLS resistant due to the ermA [TR] gene. Both MLS resistant strains were cultured from blood and belonged to T28 serotype. Multilocus sequence typing (MLST) showed that all M77 isolates belonged to sequence type 63. We conclude that 17 M77 GAS collected in the Netherlands in a 2-y period were associated with invasive disease and belonged to the same clonal complex. Since only 12% carried the ermA [TR] resistance gene, it is very likely that the gene has been acquired by horizontal transmission rather than from spread of a resistant circulating clone.

Adult↗

PubMed2005

Genetic analysis and attribution of microbial forensics evidence.

Because of the availability of pathogenic microorganisms and the relatively low cost of preparing and disseminating bioweapons, there is a continuing threat of biocrime and bioterrorism. Thus, enhanced capabilities are needed that enable the full and robust forensic exploitation and interpretation of microbial evidence from acts of bioterrorism or biocrimes. To respond to the need, greater resources and efforts are being applied to the burgeoning field of microbial forensics. Microbial forensics focuses on the characterization, analysis and interpretation of evidence for attributional purposes from a bioterrorism act, biocrime, hoax or inadvertent agent release. To enhance attribution capabilities, a major component of microbial forensics is the analysis of nucleic acids to associate or eliminate putative samples. The degree that attribution can be addressed depends on the context of the case, the available knowledge of the genetics, phylogeny, and ecology of the target microorganism, and technologies applied. The types of genetic markers and features that can impact statistical inferences of microbial forensic evidence include: single nucleotide polymorphisms, repetitive sequences, insertions and deletions, mobile elements, pathogenicity islands, virulence and resistance genes, house keeping genes, structural genes, whole genome sequences, asexual and sexual reproduction, horizontal gene transfer, conjugation, transduction, lysogeny, gene conversion, recombination, gene duplication, rearrangements, and mutational hotspots. Nucleic acid based typing technologies include: PCR, real-time PCR, MLST, MLVA, whole genome sequencing, and microarrays.

Bacterial Typing Techniques↗

PubMed2001

The evolution of pandemic clones of methicillin-resistant Staphylococcus aureus: identification of two ancestral genetic backgrounds and the associated mec elements.

Previous surveillance studies carried out by our laboratories, primarily in Southern and Eastern Europe, Latin America, and the United States, have characterized 3,067 methicillin-resistant Staphylococcus aureus (MRSA) hospital isolates by a combination of molecular typing methods. Nearly 70% of these isolates could be classified into five clonal types showing extensive geographic spread. Representative isolates of these clonal types were now reexamined for their genetic relatedness by multilocus sequence typing (MLST) and by sequencing the polymorphic region of protein A (spaA typing), and also for the type of the Staphylococcal Chromosomal Cassette (SCCmec) resident in the bacteria. Three of the previously classified clonal types (Iberian, Brazilian, and Hungarian clones) shared a common or closely related genetic background A, which was the same as the background of the earliest European isolates of MRSA from England and Denmark. The Pediatric and New York/Japan clones belonged to a completely different genetic background B. The three recently described SCCmec types were specifically associated with different pandemic clones: types I and III with isolates of genetic background A and type II with isolates of genetic background B. A novel SCCmec related to type I, called SCCmec type IV, was identified in some MRSA strains belonging to genetic background A as well as B. Structural variations in SCCmec types I and III were also observed. The data allow tentative identification of an evolutionary pathway for the emergence of pandemic MRSA clones and also provide evidence for the multiple, yet restricted, numbers of acquisition of the mec element by S. aureus.

Bacterial Proteins↗

PubMed2002

Clonal and antibiotic resistance profiles of methicillin-resistant Staphylococcus aureus (MRSA) from a Portuguese hospital over time.

Methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered from a general hospital in Oporto, Portugal, during two periods (1992-1993 and 1996-2000) were characterized by pulsed-field gel electrophoresis (PFGE) of SmaI fragments, and by hybridization of ClaI digests with mecA and Tn554 probes, discriminating the isolates in mecA::Tn554::PFGE genotypes. In addition, a representative sample of the defined genotypes was characterized by multilocus sequence typing (MLST) and SCCmec (staphylococcal cassette chromosome mec) typing, generating the corresponding ST-SCCmec types. In 1992-1993, 77% of MRSA belonged to the Iberian clone (genotype I::E::A or ST247-IA). In 1996-2000, the frequency of this clone decreased to 19% and the majority (69%) of the isolates belonged to another international clone, the Brazilian MRSA (genotype XI::B::B or ST239-IIIA). Trimethoprim/sulfamethoxazole (SXT) was confirmed to be an important phenotypic marker to distinguish the Iberian (SXT-susceptible) and the Brazilian (SXT-resistant) clones in MRSA isolates from Portugal. Our observations document major shifts in the dominant MRSA clonal types that occurred in this hospital since 1992, suggesting a selective advantage of the Brazilian relatively to the Iberian clone. In addition to these two MRSA clones that are the most frequent in Portuguese hospitals since the early 1990s, sporadic MRSA clones (representing 14% of the total) were identified and characterized.

Anti-Bacterial Agents↗

PubMed2003

Clonal analysis of Streptococcus pneumoniae nonsusceptible to penicillin at day-care centers with index cases, in a region with low incidence of resistance: emergence of an invasive type 35B clone among carriers.

The nasopharyngeal carriage rate of potential respiratory pathogens was studied in 36 index children with a pneumococci nonsusceptible to penicillin (PNSP), in 595 healthy children, and in 123 personnel at 16 day-care centers (DCCs) with index cases in the Stockholm area, an urban area with a low incidence of antibiotic resistant pneumococci, during the winter of 1997-1998. The spread and clonality of PNSP, Haemophilus influenzae and Moraxella catarrhalis, were studied by analyzing antibiotic susceptibility and serotype, and for PSNP also by using pulsed-field electrophoresis (PFGE) and multilocus sequence typing (MLST). In contrast to the low carriage rate found among the adult contacts (2%), 40% of the children harbored pneumococci, of which 20% were PNSP. Nasopharyngeal colonization decreased with age. The 49 PNSP isolates consisted of 20 clones, of which 10 could be identified in more than one child attending the same or different DCCs. In five DCCs, we observed a spread of PNSP from the index case. A novel PNSP clone of type 35B, found to cause invasive disease in several states in the United States, was found to emerge among several carriers at two DCCs . A high proportion of PNSP isolates were multiresistant to antibiotics (34%), which has implications for treatment regimens, even in a country like Sweden where the proportion of PNSP currently is low (3-4%). One PNSP clone of type 9V found among the carriers, has been shown to cause invasive disease in Sweden as well as in other countries, suggesting that one reason for the occurrence of invasive PNSP clones may be their ability to colonize and spread among healthy carriers. Other internationally spread antibiotic resistant pneumococcal clones found were of types 9V, 19F, and 23F.

Anti-Bacterial Agents↗

PubMed2003

Identification of multidrug-resistant Streptococcus pneumoniae strains isolated in Poland by multilocus sequence typing.

Multilocus sequence typing (MLST) of 35 isolates of multidrug-resistant Streptococcus pneumoniae recovered in Poland during 1995-1996 distinguished 10 different sequence types (ST). The majority of the isolates were assigned to two Polish clones of serotypes 6B and 23F, although the international clones, Spain23F-1 and Spain9V-3, were also identified. Similar results were obtained using pulsed-field gel electrophoresis (PFGE), providing a direct comparison of these two typing methods.

Bacterial Typing Techniques↗

PubMed2003

Molecular characterization of erythromycin-resistant clinical isolates of the four major antimicrobial-resistant Spanish clones of Streptococcus pneumoniae (Spain23F-1, Spain6B-2, Spain9V-3, and Spain14-5).

Erythromycin resistance and the characterization of the corresponding determinants of resistance were studied in clinical Streptococcus pneumoniae isolates belonging to the four major multiresistant pneumococcal Spanish clones (ermB and mefA genes for the Spain23F-1, Spain9V-3, serotype 14 variant of the Spain9V-3 and Spain14-5 clones and ermB gene for the Spain6B-2 clone). These isolates were confirmed as major clones by pulsed-field gel electrophoresis (PFGE), BOX-PCR, and multilocus sequence typing (MLST). The spread and prevalence of these erythromycin-resistant variants of the Spanish clones in an area of the north of Spain were dissimilar-low for the Spain9V-3 clone (5.8% among the isolates belonging to this clone, including isolates of the serotype 14 variant) and very frequent for the Spain14-5 clone (91.7%).

Anti-Bacterial Agents↗

PubMed2004

Clonal types and multidrug resistance patterns of methicillin-resistant Staphylococcus aureus (MRSA) recovered in Italy during the 1990s.

A large number (272) of methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered from Italian hospitals during the early and late 1990s were characterized for multidrug resistance pattern and clonal type using a combination of genotyping methods, including pulsed-field gel electrophoresis (PFGE), spaA typing, multilocus sequence typing (MLST), determination of SCC mec type, and hybridization pattern with Tn 554. The majority of MRSA belonged to four genetic lineages: the pandemic Iberian and Brazilian clones, and two unique clonal types-the "Italian" and "Rome" clones of MRSA. The Italian clone carried the SCC mec type I in the genetic background of ST228, which is a double-locus variant of the sequence type of the multidrug-resistant New York/Japanese clone (ST5). The properties of the Rome clone showed several striking similarities to those of the Archaic clone of MRSA that was dominant among MRSA isolates in the mid-1960s to 1970s, but has not been detected since then in recent global surveillance studies.

Amino Acid Sequence↗

PubMed2004

Identification of a variant "Rome clone" of methicillin-resistant Staphylococcus aureus with decreased susceptibility to vancomycin, responsible for an outbreak in an intensive care unit.

We describe the identification of a variant of the "Rome clone" of methicillin-resistant Staphylococcus aureus (MRSA), responsible for an outbreak involving 5 patients in a Cardiac Surgery Intensive Care Unit (CS-ICU) of a tertiary-care University Hospital in Rome. All strains isolated from patients and from nasal swabs obtained from four members of the CS-ICU personnel, belonged to the same identified clone. The characteristics of this clone were: (1) resistance to ampicillin, oxacillin, gentamicin, ciprofloxacin, erythromycin, clindamycin, rifampin, spectinomycin, and tetracycline; (2) vancomycin and teicoplanin MICs respectively of 2 and 4 mg/L; (3) heteroresistant subpopulations in the presence of 4 and 6 mg/L of vancomycin (10(-3) and 10(-5), respectively); (4) clonal type I::J::C determined following an established protocol (mec A::Tn 554 ::PFGE); (5) sequence type ST247 (3-3-1-12-4-4-16), obtained by multilocus sequence typing (MLST); and (6) the staphylococcal cassette chromosome mec (SCC) IA, obtained by multiplex PCR method. This new strain had different characteristics from the epidemic clone circulating in the same hospital from 1997 and designed "Rome clone," which was susceptible to erythromycin, clindamycin, and spectinomycin and belonged to the II::NH::C genetic background. A high genetic similarity between this Rome clone and the previously classified Archaic and Iberian clones was found, because they shared the same allelic profile (ST247), probably originating from the same S. aureus ancestor of the Iberian MRSA strains. Therefore, the strains responsible for the outbreak, with vancomycin MICs 2-4 mg/L, are variant clones, showing the genotype of the "Rome clone," the ST247 in association with SCC mec type IA (ST247-MRSA-IA), and are characterized by a uniform susceptibility to fosfomycin.

Anti-Bacterial Agents↗

PubMed2006

Canadian listeriosis reference service.

Listeria monocytogenes, a psychrotrophic organism capable of growing at refrigeration temperatures, is of major concern in extended shelf life, refrigerated foods. Considering that as much as 80-90% of human listeriosis cases are linked to the ingestion of contaminated food, human cases are predominantly seen in high-risk individuals, including organ-transplant recipients, patients with AIDS and HIV-infected individuals, pregnant women, cancer patients, and the elderly. In 2001, the Canadian Listeriosis Reference Service (LRS) was created by the Bureau of Microbial Hazards (Health Canada) and the National Microbiology Laboratory (now part of the Public Health Agency of Canada). Major goals of the LRS include investigation of listeriosis cases and maintenance of a national collection of isolates. The LRS intends to create a comprehensive molecular epidemiological database of all isolates in Canada for use as a resource for outbreak investigations, research and other microbiological investigations. The PFGE profiles are being established and stored for clinical, food, environmental, and possibly animal strains of L. monocytogenes. The LRS pursues research activities for investigation and implementation of other molecular methods for characterizing L. monocytogenes isolates. Ribotyping, Multi-locus Sequence Typing (MLST), Variable Number of Tandem Repeats (VNTR), Multi-locus virulence sequence typing (MLVA), microarray- based technologies and sequence-based typing schemes, are being investigated on selected diversity sets. The LRS has also used PFGE typing for outbreak investigations. The molecular epidemiological data, timely coordination and exchange of information should help to reduce the incidence of listeriosis in Canada. In Canada, listeriosis is not a national notifiable disease, except for the province of Quebec, where it has been since 1999. The LRS, Canadian Public Health Laboratory Network, and federal epidemiologists are currently working on making human listeriosis notifiable throughout Canada.

Canada↗