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PubMed2006

Molecular characterization of non-penicillin-susceptible Streptococcus pneumoniae in Norway.

A total of 125 non-penicillin-susceptible Streptococcus pneumoniae isolates were received at the Norwegian Institute of Public Health in the period from 1995 to 2001. The strains were tested for antimicrobial susceptibility, serotyped, and genotyped by multilocus sequence typing (MLST); and their penicillin-binding proteins (PBPs) were typed by restriction fragment length polymorphism analysis of their pbp genes. Of the 125 strains, 48 (38%) were fully resistant to penicillin and 77 (62%) were intermediately resistant to penicillin. Most of the strains resistant to penicillin were also resistant to one or several additional antibiotics. The most frequent serotypes among the non-penicillin-susceptible strains were 14, 9V, 19F, 23F, and 6B. MLST analysis showed a high degree of genetic diversity among the 119 strains tested, with a total of 74 different sequence types. Six of the 26 internationally known resistant clones were present; the Spain(9V)-3 clone was the most frequent, with 19 isolates. A total of 74 (62%) of the isolates were related to 1 of the 26 international clones. Restriction enzyme analyses of the pbp1a, pbp2b, and pbp2x genes revealed 12, 12, and 19 different patterns, respectively; and a total of 43 different PBPs types were demonstrated. Our data indicate that the non-penicillin-susceptible strains in Norway are highly diverse genetically and that limited spread of the internationally known resistant strains occurred in the country in the period examined.

Anti-Bacterial Agents↗

PubMed2006

Nonrandom distribution of Burkholderia pseudomallei clones in relation to geographical location and virulence.

Burkholderia pseudomallei is a soil-dwelling saprophyte and the causative agent of melioidosis, a life-threatening human infection. Most cases are reported from northeast Thailand and northern Australia. Using multilocus sequence typing (MLST), we have compared (i) soil and invasive isolates from northeast Thailand and (ii) invasive isolates from Thailand and Australia. A total of 266 Thai B. pseudomallei isolates were characterized (83 soil and 183 invasive). These corresponded to 123 sequence types (STs), the most abundant being ST70 (n=21), ST167 (n=15), ST54 (n=12), and ST58 (n=11). Two clusters of related STs (clonal complexes) were identified; the larger clonal complex (CC48) did not conform to a simple pattern of radial expansion from an assumed ancestor, while a second (CC70) corresponded to a simple radial expansion from ST70. Despite the large number of STs, overall nucleotide diversity was low. Of the Thai isolates, those isolated from patients with melioidosis were overrepresented in the 10 largest clones (P<0.0001). There was a significant difference in the classification index between environmental and disease isolates (P<0.001), confirming that genotypes were not distributed randomly between the two samples. MLST profiles for 158 isolates from Australia (mainly disease associated) contained a number of STs (96) similar to that seen with the Thai invasive isolates, but no ST was found in both populations. There were also differences in diversity and allele frequency distribution between the two populations. This analysis reveals strong genetic differentiation on the basis of geographical isolation and a significant differentiation on the basis of virulence potential.

Australia↗

PubMed2006

Use of a single-nucleotide polymorphism genotyping system to demonstrate the unique epidemiology of methicillin-resistant Staphylococcus aureus in remote aboriginal communities.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) has emerged as a major public health problem in Australia, as in many other parts of the world. High rates of CA-MRSA skin and soft tissue infection have been reported from Aboriginal communities. We used a single-nucleotide polymorphism (SNP) genotyping typing system based on the multilocus sequence type (MLST) database to investigate the epidemiology of CA-MRSA and methicillin-sensitive S. aureus (MSSA) over a 12-month period in three remote Aboriginal communities of Northern Australia. This was supplemented by real-time PCR for Panton-Valentine leukocidin (PVL) genes, staphylococcal cassette chromosome mec (SCCmec) typing, and antimicrobial susceptibility testing. S. aureus was recovered from pyoderma lesions on 221 occasions and throat swabs on 44 occasions. The median monthly recovery rate of S. aureus from skin sores was 58% (interquartile range, 62 to 78%), and there was no seasonal variation. Twenty-three percent of isolates were CA-MRSA; the proportion was similar across the communities and did not vary over the study period. Erythromycin resistance was found in 47% of CA-MRSA and 21% of MSSA. SNP-based typing identified 14 different clonal complexes (cc); however, cc75 was predominant, accounting for 71% of CA-MRSA isolates. These were confirmed as ST75-like by using an additional SNP and MLST of selected isolates. All but one of the cc75 isolates had SSCmec type IV (one had type V), and all were PVL negative. Monthly tracking of SNP-based cc types showed a highly dynamic process. ST75-MRSA-IV appears to be unique to the region and probably evolved de novo in remote Aboriginal communities.

Australia↗

PubMed2006

Presence of pathogenicity island genes in Enterococcus faecalis isolates from pigs in Denmark.

Enterococcus faecalis isolates of porcine origin were screened for the presence of a previously identified pathogenicity island (PAI). By using the esp gene as a genetic marker for the presence of this PAI, 9 esp-positive and 10 esp-negative isolates of porcine origin were investigated by use of a designed oligonucleotide array. The results indicated the clustering of esp-positive strains by multilocus sequence typing (MLST), but surprisingly, all strains investigated contained parts of the PAI. None of the strains of animal origin investigated belonged to previously identified MLST complex 2, where most isolates from patients cluster. Five of the nine esp-positive E. faecalis isolates of animal origin belonged to the same PAI complex as human isolate MMH594 but differed in their sequence types, which strongly indicates the horizontal transfer of the PAI between enterococci of porcine and human origin.

Animals↗

PubMed2006

Antibiotic-resistant invasive pneumococcal clones in Italy.

A total of 105 multiple-antibiotic-resistant invasive pneumococcal isolates recovered in Italy from 2001 to 2003 were genetically characterized. Of these, 40 were penicillin-nonsusceptible (PNSSP) and 65 were penicillin-susceptible (PSSP) Streptococcus pneumoniae strains. Among the PNSSP isolates, 8 and 11 different restriction profiles were obtained for the pbp2b and pbp2x genes, respectively. Clonal groups were established on the basis of analysis of both pulsed-field gel electrophoresis (PFGE) types and multilocus sequence typing (MLST). Several international clones, such as Spain(23F)-1/ST81, Spain(6B)-2/ST90, Spain(9V)-3/ST156, and Sweden(15A)-25/ST63 [corrected] were identified among the PNSSP isolates. Other, smaller clones, such as the minor Spanish 19F clone/ST88 and Denmark(14)-32/ST230, were also found. Among the PSSP isolates, clones related to England(14)-9/ST9, Greece(6B)-22/ST273, and Portugal(19F)-21/ST177 were found. In addition, two large clones comprised nonvaccine serotypes. One, comprising serotype 3 isolates, corresponded to the clone Netherlands(3)-31/ST180; the other, comprising serotype 15B/C isolates, ST474, was not related to any previously described clone. Two small clusters related to the newly described clones Greece(21)-30/ST193 and Netherlands(15B)-37/ST199 included isolates with unrelated PFGE profiles. An unusual finding was the inability to obtain the MLST allelic profile for an isolate of serotype 19A, belonging to the Sweden(15A)-25/ST63 [corrected] clone, due to a large deletion of the xpt gene. Capsular switching was observed among both PNSSP and PSSP isolates and involved also serotypes not included in the 7-valent pneumococcal conjugate vaccine (PCV7), such as serotypes 15B/C and 19A. Since antibiotic-resistant nonvaccine serotype clones are present in Italy, continuous monitoring of pneumococcal epidemiology should be carried out in the PCV7 era.

Anti-Bacterial Agents↗

PubMed2006

Probe hybridization array typing: a binary typing method for Escherichia coli.

The ability to distinguish between Escherichia coli strains is critical for outbreak investigations. Binary typing, based on the presence or absence of genetic material, provides a high-throughput alternative to gel- and PCR-based typing techniques that generate complex banding patterns and lack uniform interpretation criteria. We developed, validated, and determined the discriminatory power of an E. coli binary typing method, probe hybridization array typing (PHAT). In PHAT, the absence or presence of genetic material is identified by using DNA hybridization to produce a reproducible and portable fingerprint for each genome. PHAT probes were generated from genome subtractive hybridization experiments. We PHAT typed the ECOR collection of strains from a variety of geographical locations, and 33 rectal E. coli strains selected from college-aged women with urinary tract infection. In the set of 33 human rectal strains, the discriminatory power of PHAT (98%) equaled that of multilocus sequence typing (MLST) and pulsed-field gel electrophoresis. However, for ECOR strains, which include nonhuman strains, the current set of PHAT probes was less discriminating than MLST, ribotyping, and enterobacterial repetitive intergenic consensus sequence PCR (80% versus 97, 92, and 97%, respectively). When we limited the analysis to ECOR strains of B2 and D lineage, which are associated with human infection, current PHAT probes were highly discriminatory (94%). PHAT can be applied in a high-throughput format (i.e., "library on a slide"), the discriminatory ability can be varied based on the probe set, and PHAT is readily adapted to other bacterial species with high variation in genetic content.

Adolescent↗

PubMed2006

Validation of pulsed-field gel electrophoresis and spa typing for long-term, nationwide epidemiological surveillance studies of Staphylococcus aureus infections.

Pulsed-field gel electrophoresis (PFGE) of genomic macrorestriction fragments has been used by the Belgian Reference Laboratory for Staphylococci for national hospital surveys of methicillin-resistant Staphylococcus aureus since 1992. The sequencing of the polymorphic X region of the protein A gene (spa typing) offers significant advantages over PFGE in terms of speed, ease of interpretation, and exportability. To validate its potential use for national surveillance, we evaluated the robustness of spa typing compared with that of PFGE based on a collection of 217 S. aureus strains representative of the Belgian S. aureus epidemiology during the last 13 years. spa typing and PFGE both showed high discriminatory power (discriminatory indexes of 0.98 and 0.96, respectively) and achieved high concordance (95.9%) in type classification. Both methods also showed good concordance with multilocus sequence typing (MLST) (95.5%). However, we observed occasional "violations" of MLST clonal complex assignment by spa typing. Our results suggest that both PFGE and spa typing are reliable methods for long-term, nationwide epidemiological surveillance studies. We suggest that spa typing, which is a single-locus-based method, should preferably be used in combination with additional markers, such as staphylococcal cassette chromosome mec typing or resistance or virulence gene detection.

Anti-Bacterial Agents↗

PubMed2006

Vibrio cholerae strain typing and phylogeny study based on simple sequence repeats.

Vibrio cholerae is the etiological agent of cholera. Its natural reservoir is the aquatic environment. To date, practical typing of V. cholerae is mainly serological and requires about 200 antisera. Simple sequence repeats (SSR), also termed VNTR (for variable number of tandem repeats), provide a source of high genomic polymorphism used in bacterial typing. Here we describe an SSR-based typing method that combines the variation in highly mutable SSR loci, with that of shorter, relatively more stable mononucleotide repeat (MNR) loci, for accurate and rapid typing of V. cholerae. In silico screening of the V. cholerae genome revealed thousands of perfect SSR tracts with an average frequency of one SSR every 152 bp. A panel of 32 V. cholerae strains, representing both clinical and environmental isolates, was tested for polymorphism in SSR loci. Two strategies were applied to identify SSR variation: polymorphism of SSR tracts longer than 12 bp (L-SSR) assessed by capillary fragment-size analysis and MNR polymorphism assessed by sequencing. The nine L-SSR loci tested were all polymorphic, displaying 2 to 13 alleles per locus. Sequence analysis of eight MNR-containing loci (MNR-multilocus sequence typing [MLST]) provided information on both variations in the MNR tract itself, and single nucleotide polymorphism (SNP) in their flanking sequences. Phylogenetic analysis of the combined SSR data showed a clear discrimination between the clinical strains belonging to O1 and O139 serogroups, and the environmental isolates. Furthermore, discrimination between 27 strains of the 32 strains was achieved. SSR-based typing methods combining L-SSR and MNR-MLST were found to be efficient for V. cholerae typing.

Bacterial Typing Techniques↗

PubMed2006

Improved multilocus sequence typing scheme for Staphylococcus epidermidis.

We evaluated three multilocus sequence typing (MLST) schemes for Staphylococcus epidermidis and selected the seven most discriminatory loci for the formation of a new, more powerful MLST scheme. This improved scheme gave 31 sequence types (STs) and 5 clonal complexes (CCs), whereas the other schemes delineate 16 to 24 STs and 1 to 3 CCs.

Algorithms↗

PubMed2007

Use of outer surface protein repeat regions for improved genotyping of Staphylococcus epidermidis.

Staphylococcus epidermidis is an important nosocomial pathogen, but little is known of its epidemiology. Accurate, reproducible typing systems would greatly improve epidemiologic investigations of S. epidermidis. The sequence-based typing technique most recently evaluated, multilocus sequence typing (MLST), often lacks discrimination and can be expensive. PCR and sequence-based analyses of the serine-aspartate repeat region of sdrG (Fbe) and the repeat region of the accumulation-associated protein gene (aap) were evaluated for the ability to discriminate among previously well-characterized S. epidermidis clinical isolates. Forty-eight strains were investigated, with sdrG found in 100% and aap found in 79% of all strains tested. Both genes demonstrated PCR product size and nucleotide sequence variation. Each system by itself gave an index of discrimination similar in value to that of MLST (0.924 and 0.953 compared to 0.96), but discrimination was further improved when combinations of the three systems were used. We conclude that typing systems using amino acid and nucleotide repeat regions of the S. epidermidis surface proteins SdrG and Aap show promise as typing tools and should be investigated using a larger panel of clinically relevant isolates.

Amino Acid Sequence↗

PubMed2006

Genetically diverse group A streptococci from children in far-western Nepal share high genetic relatedness with isolates from other countries.

The genetic diversity of group A streptococci (GAS) throughout much of the world has not been adequately explored. To assess genetic variation among GAS in western Nepal, 120 noninvasive GAS, collected from eight different villages, were genetically characterized using emm typing, sof sequencing, and multilocus sequence typing (MLST). A high level of genetic diversity was observed among these isolates, with 51 genotypes based upon 51 multilocus sequence types (STs), 45 emm sequence types, and 28 sof sequence types. On the basis of shared ST-emm and sof-emm associations, 40 of the 51 genotypes were identical or highly related to genotypes characterized from locations outside of Nepal, even though most of the emm sequence and clonal types are rare among GAS within the United States. When analyzing all known STs highly related to Nepal STs, only one example of similar STs shared between a sof PCR-positive strain and a sof PCR-negative strain was observed. Since previous data indicate free exchange of MLST loci between sof-positive and sof-negative strains, there is possibly selection against the expansion of subclones resulting from horizontal transfers of sof or emm genes between sof-positive and sof-negative strains. All 45 emm types encountered in Nepal have also been documented from other countries. These data, together with data encompassing the past decade of emm type surveillance, support the possibility that most existing GAS emm types have been discovered. Similarly, since most (40/51) strain types were highly related to strains found elsewhere, it is likely that a major fraction of the existing GAS clonal complexes have been discovered.

Antigens, Bacterial↗

PubMed2000

The 1998 Senegal epidemic of meningitis was due to the clonal expansion of A:4:P1.9, clone III-1, sequence type 5 Neisseria meningitidis strains.

Between January and April 1998, a meningitis outbreak due to serogroup A meningococcus took place in Senegal. The outbreak began in Gandiaye, 165 km to the east of Dakar, and progressed towards the towns of Gossas, Niakkhar, Guinguineo, Fatik, Foundiougne, Dioffior, Sokone, Kaolack, and Nioro. At the same time, the outbreak reached regions of Kaffrine, Koungheul, and Tambacounda in the east of Senegal. A total of 1,350 cases and 200 deaths were reported. The WHO Collaborating Center in Marseilles received 24 strains for analysis. All were serogroup A Neisseria meningitidis, type 4 and subtype P1.9. Multilocus enzyme electrophoresis, performed by Institut Pasteur Paris, showed that the strains belonged to clone III-1. DNA restriction fragments generated by endonuclease BglII and analyzed by pulsed-field gel electrophoresis showed 24 indistinguishable fingerprint patterns similar to those of meningococcus strains isolated from African outbreaks since 1988. Three strains were studied by multilocus sequence typing (MLST) with seven loci. The comparison between sequences and existing alleles on the MLST website () allowed us to assign these strains to sequence type 5 (ST5), as their sequences were identical to the consensus at seven loci. All 24 strains were susceptible to penicillin, amoxicillin, chloramphenicol, and rifampin. Subgroup III is finishing its spread towards west of the meningitis belt of Africa. To our knowledge, this is the first time subgroup III, and more precisely ST5, strains are reported as being responsible for a meningitis outbreak in Senegal.

Bacterial Typing Techniques↗

PubMed2000

Fluorescent amplified-fragment length polymorphism genotyping of Neisseria meningitidis identifies clones associated with invasive disease.

Fluorescent amplified-fragment length polymorphism (FAFLP), a genotyping technique with phylogenetic significance, was applied to 123 isolates of Neisseria meningitidis. Nine of these were from an outbreak in a British university; 9 were from a recent outbreak in Pontypridd, Glamorgan; 15 were from sporadic cases of meningococcal disease; 26 were from the National Collection of Type Cultures; 58 were carrier isolates from Ironville, Derbyshire; 1 was a disease isolate from Ironville; and five were representatives of invasive clones of N. meningitidis. FAFLP analysis results were compared with previously published multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) results. FAFLP was able to identify hypervirulent, hyperendemic lineages (invasive clones) of N. meningitidis as well as did MLST. PFGE did not discriminate between two strains from the outbreak that were classified as similar but distinct by FAFLP. The results suggest that high resolution of N. meningitidis for outbreak and other epidemiological analyses is more cost efficient by FAFLP than by sequencing procedures.

Bacterial Typing Techniques↗

PubMed2001

Multilocus sequence typing system for Campylobacter jejuni.

The gram-negative bacterium Campylobacter jejuni has extensive reservoirs in livestock and the environment and is a frequent cause of gastroenteritis in humans. To date, the lack of (i) methods suitable for population genetic analysis and (ii) a universally accepted nomenclature has hindered studies of the epidemiology and population biology of this organism. Here, a multilocus sequence typing (MLST) system for this organism is described, which exploits the genetic variation present in seven housekeeping loci to determine the genetic relationships among isolates. The MLST system was established using 194 C. jejuni isolates of diverse origins, from humans, animals, and the environment. The allelic profiles, or sequence types (STs), of these isolates were deposited on the Internet (http://mlst.zoo.ox.ac.uk), forming a virtual isolate collection which could be continually expanded. These data indicated that C. jejuni is genetically diverse, with a weakly clonal population structure, and that intra- and interspecies horizontal genetic exchange was common. Of the 155 STs observed, 51 (26% of the isolate collection) were unique, with the remainder of the collection being categorized into 11 lineages or clonal complexes of related STs with between 2 and 56 members. In some cases membership in a given lineage or ST correlated with the possession of a particular Penner HS serotype. Application of this approach to further isolate collections will enable an integrated global picture of C. jejuni epidemiology to be established and will permit more detailed studies of the population genetics of this organism.

Alleles↗

PubMed2002

Determining the genetic structure of the natural population of Staphylococcus aureus: a comparison of multilocus sequence typing with pulsed-field gel electrophoresis, randomly amplified polymorphic DNA analysis, and phage typing.

We used a sample of Staphylococcus aureus strains that are carried by humans and that are representative of the natural population of S. aureus strains in order to assess the value of multilocus sequence typing (MLST), pulsed-field gel electrophoresis, randomly amplified polymorphic DNA analysis, and phage typing as epidemiological tools. Only MLST was able to define clonal complexes unambiguously. All DNA-based typing approaches achieved a high degree of agreement, implying phylogenetic concordance, but predicted epidemiological associations with variable accuracy.

Bacterial Typing Techniques↗

PubMed2002

Automated ribotyping of vancomycin-resistant Enterococcus faecium isolates.

Vancomycin-resistant Enterococcus faecium (VREF) strains represent an important threat in hospital infections in the United States and are found at high frequencies in both the community and farm animals in Europe. We evaluated automated ribotyping for interlaboratory reproducibility by using the restriction enzymes EcoRI and BamHI and compared ribotyping to both amplification of fragment length polymorphism (AFLP) analysis and multilocus sequence typing (MLST) to assess its discriminatory power and capacity for the identification of epidemiologically important strains. Of 19 (EcoRI) and 16 (BamHI) isolates tested in duplicate in two laboratories, 18 (95%) and 16 (100%), respectively, showed reproducible ribotypes. These high reproducibility rates were obtained only after manual refinement of the automated fingerprint analysis. A group of 49 VREF strains initially selected to represent 32 distinct AFLP types were separated into 28 EcoRI ribotypes, 25 BamHI ribotypes, and 28 sequence types. Ribotyping with EcoRI and BamHI was able to discern the host-specific genogroups recently disclosed by AFLP typing and MLST and to distinguish most strains containing the esp gene, a marker specific for strains causing hospital outbreaks. An expandable ribotype identification library was created. We recommend EcoRI as the enzyme of choice for automated ribotyping of VREF strains. Given the high level of discrimination of VREF strains, the high rate of interlaboratory reproducibility, and the potential for the identification of epidemiologically important genotypes, automated ribotyping appears to be a very valuable approach for characterizing VREF strains.

Alleles↗

PubMed2003

Genetic diversity of Neisseria gonorrhoeae housekeeping genes.

Molecular typing of Neisseria gonorrhoeae strains is an important tool for epidemiological studies of gonococcal infection and transmission. The recently developed multilocus sequence typing (MLST) method is based on the genetic variation among housekeeping genes. As a preliminary investigation for the development of such a method, we characterized the genetic diversity at 18 gonococcal housekeeping gene loci. Approximately 17,500 nucleotides, spanning 18 loci, were sequenced from 24 isolates. Including strain FA 1090, which has been fully sequenced, and three unique glnA sequences from GenBank, the number of alleles identified for the 18 loci ranged from 2 to 18, with a mean of 8.3 alleles per locus. The majority of polymorphic sites were distributed randomly along the genes, consistent with evolution of DNA sequences by point mutation. In addition, several examples of clustered mutations and insertions or deletions were detected, which most likely occurred by recombinational events. While purifying selection is the dominant force driving the evolution of these housekeeping genes, positive selection also appeared to operate on the abcZ and gpdh loci. The 25 completely characterized strains each had a unique allelic profile with as few as three loci (pilA, abcZ, and pip or pgi2). Molecular typing based on the allelic profile of housekeeping genes resolved the isolates better than either porB nucleotide sequencing or typing of the opa gene. The allelic profiles for the pilA, abcZ, and serC loci of paired strains from 16 sexual contacts were identical. A potential MLST for N. gonorrhoeae, based on approximately 500- to 600-bp gene fragments of seven housekeeping gene loci, would include the pilA, abcZ, serC, glnA, gdh, gnd, and pip loci.

Base Sequence↗

PubMed2003

Characterization of the natural population of Bartonella henselae by multilocus sequence typing.

Investigations of the population genetics of Bartonella henselae have demonstrated a high level of diversity among strains, and the delineation of isolates into one of two subtypes, type I (Houston) and type II (Marseille), represented by specific 16S ribosomal DNA (rDNA) sequences, has long been considered the most significant genotypic division within the species. This belief is challenged by recent work suggesting a role for horizontal gene exchange in generating intraspecies diversity. We attempted to resolve this issue and extend exploration of the population structure of B. henselae by using multilocus sequence typing (MLST) to examine the distribution of polymorphisms within nine different genes in a sample of 37 human and feline isolates. MLST distinguished seven sequence types (STs) that resolved into three distinct lineages, suggesting a clonal population structure for the species, and support for these divisions was obtained by macrorestriction analysis using pulsed-field gel electrophoresis. The distribution of STs among isolates recovered from human infections was not random, and such isolates were significantly more often associated with one particular ST, lending further support to the suggestion that specific genotypes contribute disproportionately to the disease burden in humans. All but one isolate lay on lineages that bore the representative strain of either the Houston or Marseille subtype. However, the distribution of the two 16S rDNA alleles among the isolates was not entirely congruent with their lineage allocations, indicating that this is not a sensitive marker of the clonal divisions within the species. The inheritances of several of the genes studied could not be reconciled with one another, providing further evidence of horizontal gene transfer among B. henselae strains and suggesting that recombination has a role in shaping the genetic character of bartonellae.

Animals↗