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Results for “Multilocus sequence typing”

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PubMed2006

Sequence analysis of Enterococcus faecium strain 10/96A (VanD4), the original vancomycin-resistant E. faecium strain in Brazil.

Enterococcus faecium strain 10/96A (VanD4) was the first vancomycin-resistant enterococcus (VRE) isolated in Brazil. Subsequent Brazilian VRE strains have all had the VanA phenotype. Multilocus sequence typing showed that strain 10/96A was isolated sporadically, has a unique sequence type (ST 281), and was not the progenitor of the VRE strains isolated from hospital outbreaks in Brazil.

Brazil↗

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

Staphylococcus aureus genotyping using novel real-time PCR formats.

One approach to microbial genotyping is to make use of sets of single-nucleotide polymorphisms (SNPs) in combination with binary markers. Here we report the modification and automation of a SNP-plus-binary-marker-based approach to the genotyping of Staphylococcus aureus and its application to 391 S. aureus isolates from southeast Queensland, Australia. The SNPs used were arcC210, tpi243, arcC162, gmk318, pta294, tpi36, tpi241, and pta383. These provide a Simpson's index of diversity (D) of 0.95 with respect to the S. aureus multilocus sequence typing database and define 61 genotypes and the major clonal complexes. The binary markers used were pvl, cna, sdrE, pT181, and pUB110. Two novel real-time PCR formats for interrogating these markers were compared. One of these makes use of "light upon extension" (LUX) primers and biplexed reactions, while the other is a streamlined modification of kinetic PCR using SYBR green. The latter format proved to be more robust. In addition, automated methods for DNA template preparation, reaction setup, and data analysis were developed. A single SNP-based method for ST-93 (Queensland clone) identification was also devised. The genotyping revealed the numerical importance of the "South West Pacific" and "Queensland" community-acquired methicillin-resistant S. aureus (MRSA) clones and the clonal complex 239 "Aus-1/Aus-2" hospital-associated MRSA. There was a strong association between the community-acquired clones and pvl.

Bacterial Typing Techniques↗

PubMed2006

Characteristics of Staphylococcus aureus strains isolated in Poland in 1996 to 2004 that were deficient in species-specific proteins.

One hundred seventy Staphylococcus aureus isolates, collected in 1996 to 2004, were reidentified by phenotypic and genotypic methods. One hundred ten of these (65%) were confirmed, as previously denoted, to be clumping factor (CF)- or free coagulase-deficient S. aureus, based on their phenotype. Based on the CF or coagulase production, three groups of phenotypically deficient S. aureus isolates were distinguished. Group 1 encompassed CF-positive and coagulase-deficient isolates, group 2 consisted of CF-deficient and coagulase-positive isolates, and group 3 included isolates that were CF positive, had delayed coagulase activity, and were deficient in other species-specific features. All investigated strains harbored the clfA, clfB, coa, spa, and nuc genes, but the presence of their products was not detected by the phenotypic methods. Glycopeptide susceptibility testing showed that 26 isolates (23.6%) were hetero-glycopeptide-intermediate S. aureus(hGISA) or hetero-teicoplanin-intermediate S. aureus (hTISA), based on the population analysis profile. The relatedness of the isolates was evaluated by multiple-locus variable number of tandem repeats analysis, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing. The phenotypically deficient S. aureus isolates were classified into PFGE types B (ST239-III) and D (ST246-IA) and were related to the common clones, Hungarian and Iberian, respectively, which have been widely disseminated in Poland and globally. The simultaneous occurrence of hGISA/hTISA and the CF-deficient phenotypes was found for 62.1% of isolates belonging to group 2. The majority of these isolates were assigned to the Iberian clone (PFGE type D; ST247-IA). An association between the defect in coagulase and that in thermonuclease production was observed, which concerned 59.2% of isolates of group 1. The majority of these isolates belonged to the Hungarian clone (PFGE type B; ST239-III).

Bacterial Typing Techniques↗

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

Phylogenetic comparisons reveal multiple acquisitions of the toxin genes by enterotoxigenic Escherichia coli strains of different evolutionary lineages.

Escherichia coli is a diverse bacterial species which is widely distributed in the environment but also exists as a commensal and pathogen of different host species. Human intestinal pathogenic E. coli causes over 160 million cases of diarrhea and an estimated 1 million deaths per year. The majority of deaths are attributable to one pathovar of E. coli, namely, enterotoxigenic E. coli. The pathogenesis of enterotoxigenic E. coli is dependent on the production of a colonization factor to promote adhesion to the intestinal epithelium and the elaboration of heat-labile or heat-stable toxins which induce a secretory diarrhea. Despite the high morbidity and mortality associated with enterotoxigenic E. coli infection, little is known of the genetic background of this global pathogen. Here we demonstrate by multilocus sequence typing that enterotoxigenic E. coli isolates are present in all phylogenetic lineages of E. coli, indicating that acquisition of the toxin genes may be sufficient to generate an enterotoxigenic E. coli strain. In addition, screening of diarrheal isolates for the presence of additional genes previously associated with the virulence of enterotoxigenic E. coli revealed that they were not abundant. These observations have significant implications for disease epidemiology and for the design of effective vaccines.

Bacterial Toxins↗

PubMed2006

Molecular characterization of nontypeable group B streptococcus.

Traditionally, the capsular polysaccharide (CPS) antigen has been used to distinguish between the nine known serotypes of group B streptococcus (GBS) by classical antibody-antigen reactions. In this study, we used PCR for all CPSs and selected protein antigens, multilocus sequencing typing (MLST), and pulsed-field gel electrophoresis (PFGE) to molecularly characterize 92 clinical isolates identified as nontypeable (NT) by CPS-specific antibody-antigen reactivity. The PCR and MLST were performed on blinded, randomly numbered isolates. All isolates contained the cfb gene coding for CAMP factor. While most (56.5%) contained a single CPS-specific gene, 40 isolates contained either two or three CPS-specific genes. Type V CPS-specific gene was present in 66% of the isolates, and all serotypes except types IV, VII, and VIII were represented. Most (44.5%) of the isolates contained a single protein antigen gene (bca, bac, rib, alp1, or alp3), and the remaining isolates had multiple protein antigen genes. Of the 61 isolates that had the V CPS-specific gene, 48 (78.6%) had the alp3 gene. PFGE analysis classified the isolates into 21 profile groups, while MLST analysis divided the isolates into 16 sequence types. Forty-two (69%) of 61 isolates with the V CPS-specific gene were in PFGE profile group 4; 41 of these 42 were sequence type 1 by MLST. These data shed new light on the antigenic complexity of NT GBS isolates, information that can be valuable in the formulation of an effective GBS vaccine.

Animals↗

PubMed1998

Necessity of molecular techniques to distinguish between Neisseria meningitidis strains isolated from patients with meningococcal disease and from their healthy contacts.

Serogroup C strains of Neisseria meningitidis were isolated from a Germany patient with severe meningococcal disease after a trip to the Czech Republic. These strains (case isolates) were characterized by classical and molecular techniques, as were other strains (carrier isolates) isolated from healthy contacts. Five of 10 carrier isolates had switched off the expression of capsular polysaccharide, as demonstrated by a serogroup-specific PCR. The two case isolates were indistinguishable by multilocus sequence typing and belonged to the ET-37 complex. The carrier isolates belonged to four different sequence types, all unrelated to that of the case strains. Pulsed-field gel electrophoresis showed that the case isolates differed from reference ET-37 complex strains from the Czech Republic and Canada as well as from all the carrier isolates. The isolate from the patient's nasopharynx was indistinguishable from the blood isolate except for a 40,000-bp chromosomal deletion that had occurred during systemic spread.

Adolescent↗

PubMed1999

The three major Spanish clones of penicillin-resistant Streptococcus pneumoniae are the most common clones recovered in recent cases of meningitis in Spain.

One hundred six isolates of Streptococcus pneumoniae recovered in Spain from patients with meningitis in 1997 and 1998 were characterized by multilocus sequence typing. A heterogeneous collection of genotypes was associated with meningitis in Spain: 65 different sequence types were resolved and, even at a genetic distance of 0.43, there were 37 distinct lineages. Thirty-eight percent of the isolates, including all isolates of serotypes 6B, 9V, 14, and 23F, were resistant to penicillin, and 24% of the isolates were members of the three major Spanish penicillin-resistant or multidrug-resistant clones of serotypes 6B, 9V, and 23F or serotype variants of these clones. These three clones (MICs, 1 to 2 microg of penicillin/ml) were the most common clones associated with pneumococcal meningitis in Spain during 1997 and 1998. Only two of the other clones associated with meningitis were penicillin resistant (MICs, 0.12 to 0.5 microg/ml). One of the two most prevalent penicillin-susceptible clones causing meningitis (serotype 3) has not been detected outside of Spain, whereas the other (serotype 18C) has been recovered from patients with meningitis in the United Kingdom, The Netherlands, and Denmark. The prevalence of meningitis caused by isolates of the three major Spanish penicillin-resistant or multiply antibiotic-resistant clones, which are now globally distributed, is disturbing and clearly establishes their ability to cause life-threatening disease.

Base Sequence↗

PubMed2000

Carried meningococci in the Czech Republic: a diverse recombining population.

Population and evolutionary analyses of pathogenic bacteria are frequently hindered by sampling strategies that concentrate on isolates from patients with invasive disease. This is especially so for the gram-negative diplococcus Neisseria meningitidis, a cause of septicemia and meningitis worldwide. Meningococcal isolate collections almost exclusively comprise organisms originating from patients with invasive meningococcal disease, although this bacterium is a commensal inhabitant of the human nasopharynx and very rarely causes pathological effects. In the present study, molecular biology-based techniques were used to establish the genetic relationships of 156 meningococci isolated from healthy young adults in the Czech Republic during 1993. None of the individuals sampled had known links to patients with invasive disease. Multilocus sequence typing (MLST) showed that the bacterial population was highly diverse, comprising 71 different sequence types (STs) which were assigned to 34 distinct complexes or lineages. Three previously identified hyperinvasive lineages were present: 26 isolates (17%) belonged to the ST-41 complex (lineage 3); 4 (2.6%) belonged to the ST-11 (electrophoretic type [ET-37]) complex, and 1 (0.6%) belonged to the ST-32 (ET-5) complex. The data were consistent with the view that most nucleotide sequence diversity resulted from the reassortment of alleles by horizontal genetic exchange.

Adolescent↗

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↗

PubMed2002

Molecular peculiarities of the lytA gene isolated from clinical pneumococcal strains that are bile insoluble.

The autolytic LytA amidase from 12 bile (deoxycholate)-insoluble streptococcal isolates (formerly classified as atypical Streptococcus pneumoniae) showing different antibiotic resistance patterns was studied. These atypical strains, which autolyze at the end of the stationary phase of growth, contain highly divergent lytA alleles (pairwise evolutionary distances of about 20%) compared to the lytA alleles of typical pneumococci. The atypical LytA amidases exhibit a peculiar deletion of two amino acids responsible for cell wall anchoring in the carboxy-terminal domain and have a reduced specific activity. These enzymes were inhibited by 1% deoxycholate but were activated by 1% Triton X-100, a detergent that could be used as an alternative diagnostic test for this kind of strain. Preparation of functional chimeric enzymes, PCR mutagenesis, and gene replacements demonstrated that the characteristic bile insolubility of these atypical strains was due to their peculiar carboxy-terminal domain and that the 2-amino-acid deletion was responsible for the inhibitory effect of deoxycholate. However, the deletion alone did not affect the specific activity of LytA. A detailed characterization of the genes encoding the 16S rRNA and SodA together with multilocus sequence typing indicated that the strains studied here are not a single clone and, although they cannot be strictly classified as typical pneumococci, they represent a quite diverse pool of organisms closely related to S. pneumoniae. The clinical importance of these findings is underlined by the role of the lytA gene in shaping the course of pneumococcal diseases. This study can also contribute to solving diagnostic problems and to understanding the evolution and pathogenic potential of species of the Streptococcus mitis group.

Alleles↗

PubMed2002

Molecular epidemiology of erythromycin resistance in Streptococcus pneumoniae isolates from blood and noninvasive sites.

Erythromycin-resistant isolates of Streptococcus pneumoniae from blood cultures and noninvasive sites were studied over a 3-year period. The prevalence of erythromycin resistance was 11.9% (19 of 160) in blood culture isolates but 4.2% (60 of 1,435) in noninvasive-site isolates. Sixty-two of the 79 resistant isolates were available for study. The M phenotype was responsible for 76% (47 of 62) of resistance, largely due to a serotype 14 clone, characterized by multilocus sequence typing as ST9, which accounted for 79% (37 of 47) of M phenotype resistance. The ST9 clone was 4.8 times more common in blood than in noninvasive sites. All M phenotype isolates were PCR positive for mef(A), but sequencing revealed that the ST9 clone possessed the mef(A) sequence commonly associated with Streptococcus pyogenes. All M phenotype isolates with this mef(A) sequence also had sequences consistent with the presence of the Tn1207.1 genetic element inserted in the celB gene. In contrast, isolates with the mef(E) sequence normally associated with S. pneumoniae contained sequences consistent with the presence of the mega insertion element. All MLS(B) isolates carried erm(B), and two isolates carried both erm(B) and mef(E). Fourteen of the 15 MLS(B) isolates were tetracycline resistant and contained tet(M). However, six M phenotype isolates of serotypes 19 (two isolates) and 23 (four isolates) were also tetracycline resistant and contained tet(M). MICs for isolates with the mef(A) sequence were significantly higher than MICs for isolates with the mef(E) sequence (P < 0.001). Thus, the ST9 clone of S. pneumoniae is a significant cause of invasive pneumococcal disease in northeast Scotland and is the single most important contributor to M phenotype erythromycin resistance.

Anti-Bacterial Agents↗

PubMed2003

Stability of serotypes during nasopharyngeal carriage of Streptococcus pneumoniae.

Serotype changes among natural isolates of Streptococcus pneumoniae are well documented and occur by recombinational exchanges at the capsular biosynthetic locus. However, the frequency with which this phenomenon occurs within the nasopharynx of children is not clear and is likely to be highest in the nasopharynx of children, who have high rates of pneumococcal carriage. A birth cohort of 100 infants was studied, and pneumococci were recovered from nasopharyngeal samples taken at monthly intervals during the first 6 months of life and then at 2-monthly intervals until the age of 2 years. Among the 1,353 nasopharyngeal samples were 523 that contained presumptive pneumococci, and three colonies from each were serotyped. A total of 333 isolates, including all isolates of differing serotypes from the same child, were characterized by multilocus sequence typing. Sixty-eight children carried multiple serotypes during the first 2 years of life. Two children carried a typeable and a nonserotypeable pneumococcus of identical genotype, and five children carried genetically indistinguishable isolates of serotypes 15B and 15C. These isolates were considered, respectively, to be due to loss of capsule expression and the known ability of serotype 15B and 15C pneumococci to interconvert by loss or gain of an acetyl group on the capsular polysaccharide. In all other cases, isolates from the same children that differed in serotype also differed in genotype, indicating the acquisition of a different pneumococcal strain rather than a change in capsular type. There was therefore no evidence in this study for any change of serotype due to recombinational replacements at the capsular locus among the pneumococci carried within the nasopharynges of the children.

Carrier State↗

PubMed2003

Molecular characterization of a strain of group a streptococcus isolated from a patient with a psoas abscess.

We report the first case of a primary group A streptococcus (GAS) psoas abscess in a 31-year-old woman. The psoas abscess was preceded by an episode of acute pharyngitis. The M-protein gene (emm) and streptolysin S structural gene (sagA) were present in the isolate, with no significant amino acid differences from previously described sequences of M1 GAS isolates. Multilocus sequence typing (MLST) showed that the isolate belonged to MLST sequence type (MLST-ST) 28, the predominant MLST-ST associated with invasive disease caused by M1 isolates.

Adult↗

PubMed2003

Geographic distribution and clonal diversity of Streptococcus pneumoniae serotype 1 isolates.

Serotype 1 pneumococci are a major cause of serious disease and have been associated with outbreaks but are rarely carried. The high attack rate and lack of coverage of this serotype by the heptavalent conjugate vaccine prompted the characterization of a geographically diverse collection of 166 serotype 1 isolates from recent cases of invasive disease. The isolates were resolved by multilocus sequence typing into 16 clones, which clustered into three major lineages with very different geographic distributions. Lineage A isolates were exclusively from Europe and North America, lineage B isolates were predominantly from Africa and Israel, and lineage C isolates were mainly from Chile. There was no clear association between the presence of individual clones within a country and the prevalence of serotype 1 disease.

Africa↗

PubMed2003

Molecular characterization of ampicillin-resistant Enterococcus faecium isolates from hospitalized patients in Norway.

The genetic relationship of 81 ampicillin-resistant and 21 ampicillin-susceptible Enterococcus faecium isolates from clinical infections and rectal screening in hospitalized patients in Norway was studied by pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP). PFGE showed 55 different banding patterns, and 65 of the isolates could be grouped into one large group. With AFLP, 46 patterns were discerned, and 74 isolates clustered in one group. In general, the isolates had a higher degree of similarity than with PFGE. The purK gene, which is one of the targets of the E. faecium multilocus sequence typing scheme, was sequenced. Eleven different purK alleles could be discerned, with the majority of isolates (n = 80) harboring allele 1. With only two exceptions, all strains carrying purK-1 clustered in the same PFGE and AFLP groups, indicating a good correlation between PFGE type, AFLP type, and purK allele. Genetic polymorphism of a 571-bp PCR fragment of the C-terminal domain of the penicillin-binding protein 5 gene (pbp5) was determined, and sequence differences were associated with the level of ampicillin resistance. This study indicates that the majority of ampicillin-resistant E. faecium strains in Norway belong to a distinct genetic lineage of closely related genotypes. Rectal and clinical isolates were generally indistinguishable, and differences in clonal distribution and allele polymorphism were found mainly between ampicillin-resistant and -susceptible isolates.

Ampicillin↗