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PubMed2004

[Characterization of Haemophilus influenzae strains using multilocus sequencing].

First results of multilocus sequence typing (MLST) of Haemophilus influenzae strains are presented. MLST of 28 H. influenzae strains isolated from patients with invasive diseases in the Czech Republic is indicative of clonal homogeneity of these strains: 22 out of 26 H. influenzae b strains tested were of the same sequence type, ST-6. Four strains were of two sequence types newly described in this study: ST-83 (3 strains) and ST-84 (1 strain). Two nontypeable H. influenzae strains were assigned to sequence types other than ST-6: ST-3 and ST-85 newly described in this study. First MLST results show ST-6 to be typical of H. influenzae b isolated from patients with invasive diseases in the Czech Republic. The sequence types newly described in this study, i.e. ST-83, ST-84 and ST-85, were submitted to the worldwide H. influenzae MLST database (http://haemophilus.mlst.net).

Bacterial Typing Techniques↗
PubMed2001

Genetic and phenotypic features of Streptococcus pyogenes strains isolated in Brazil that harbor new emm sequences.

In the present study, 37 group A Streptococcus (GAS) strains belonging to 13 new emm sequence types identified among GAS strains randomly isolated in Brazil were characterized by using phenotypic and genotypic methods. The new types were designated st204, st211, st213, st809, st833, st854, st2904, st2911, st2917, st2926, st3757, st3765, and st6735. All isolates were susceptible to the antimicrobial agents tested, except to tetracycline. They all carried the speB gene, and 94.6% produced detectable SpeB. Most strains belonging to a given emm type had similar or highly related pulsed-field gel electrophoresis profiles that were distinct from profiles of strains of another type. The other characteristics were variable from isolate to isolate, although some associations were consistently found within some emm types. Unlike the other isolates, all type st213 isolates were speA positive and produced SpeA. Strains belonging to st3765 were T6 and opacity factor (OF) negative. Individual isolates within OF-positive emm types were associated with unique sof gene sequence types, while OF-negative isolates were sof negative by PCR. This report provides information on new emm sequence types first detected in GAS isolates from a geographic area not extensively surveyed. Such data can contribute to a better understanding of the local and global dynamics of GAS populations and of the epidemiological aspects of GAS infections occurring in tropical regions.

Antigens, Bacterial↗
PubMed2005

Discrimination within phenotypically closely related definitive types of Salmonella enterica serovar typhimurium by the multiple amplification of phage locus typing technique.

Multilocus sequence typing (MLST) is a relatively new high-resolution typing system employed for epidemiological studies of bacteria, including Salmonella. Discrimination based on MLST of housekeeping genes may be problematical, due to the high identity of gene sequences of closely related Salmonella species. The presence of genomic sequences derived from stable temperate phages in Salmonella offers an alternative for MLST of Salmonella. We have used MLST of prophage loci in Salmonella enterica serovar Typhimurium to discriminate closely related isolates of serovar Typhimurium. We have compared these results to MLST of five housekeeping genes, as well as pulsed-field gel electrophoresis (PFGE). The presence or absence of prophage loci in the 73 serovar Typhimurium isolates tested, as well as allelic variation as detected by sequencing, provided greater discrimination between isolates than either MLST of housekeeping genes or PFGE. Amplification of prophage loci alone separated serovar Typhimurium isolates into 27 groups comprising multiple isolates or individual strains. Sequencing of isolates found within the clusters separated isolates even further. By contrast, PFGE could only divide the 73 isolates into five distinct groups. MLST using housekeeping genes did not provide any significant separation of isolates in comparison to amplification or MLST of prophage loci. The results demonstrate that the amplification and sequencing of prophage loci provides a high-resolution, objective method for the discrimination of closely related isolates of serovar Typhimurium. It is proposed that multiple amplification of phage locus typing may provide sufficient discrimination for epidemiological purposes without recourse to MLST.

Bacterial Proteins↗
PubMed1996

Alternative splicing in the coding region of human aromatic L-amino acid decarboxylase mRNA.

Total RNA from human neuroblastoma cells (SK-N-SH) was reverse transcribed and amplified using primers specific for aromatic L-amino acid decarboxylase (AADC). Two polymerase chain reaction (PCR) products were observed following agarose electrophoresis. Cycle sequencing of the PCR products revealed the larger fragment (414 bp) to be identical to the published human cDNA sequence (Type I). Sequencing of the smaller band (300 bp) demonstrated a form missing exon three (Type II). Both types of the mRNA were colocalized in human brain regions (gray matter and white matter) and other human tissues (liver, kidney, adipose, heart, adrenal gland and keratinocytes). The relative concentrations varied in each tissue studied but specific neuronal or non-neuronal patterns were not apparent. The study demonstrates alternative splicing within the coding region of the human AADC mRNA and the results suggest the possibility that two proteins are derived from the AADC gene in human tissues.

Aromatic-L-Amino-Acid Decarboxylases↗
PubMedDate not supplied

High throughput HLA sequence-based typing (SBT) utilizing the ABI Prism 3700 DNA Analyzer.

AIMS AND BACKGROUND: The genetic complexity of the human major histocompatibility complex (MHC) has required the development of various molecular typing methods. The purpose of this paper is to compare the results of two of these molecular methods: sequenced based typing (SBT) and polymerase chain reaction (PCR) using sequence specific primers (PCR-SSP). METHODS: The SBT method described utilizes an ABI Prism 3700 DNA Analyzer, which has been designed fro high throughput production of sequence data through highly automated operation with significant walk-away time. The ABI Prism 3700 DNA Analyzer is a 96-capillary electrophoresis instrument with the capability of running four 96-well plates black to back in a sixteen-hour period. Potentially, data from this machine can produce Class I sequences for A or B loci for 64 samples in this time frame. The SBT method encompassed exons 2, 3, and 4 with forward and reverse sequence orientation reactions using the PE Biosystems HLA-A and HLA-B Sequenced Based Typing Kits (PE Applied Biopsystems/Perkin-Elmer, Foster City, CA, USA). Most SBT methods previously employed only gather data from exons 2 and 3 which distinguishes most of the polymorphism necessary to identify the majority of alleles in the HLA region. However, in an effort to discern numerous null alleles in the HLA region, exon 4 data is also included. The PCR-SSP method utilized consists of one 96 well tray, with 95 primer mixes and one negative control, per sample designed to produce an intermediate/high resolution HLA-A, B typing. RESULTS: Data from one 96-well capillary run on the ABI Prism 3700 DNA Analyzer, which consists of results from 16 samples for HLA-A or HLA-B loci, was compared to data derived from sixteen HLA-A and HLA-B PCR-SSP typings. 75% of loci tested achieved a higher resolution HLA typing by the SBT method. DISCUSSION: The ability to provide allele level HLA typing results can have significant functional implications for the bone marrow transplant community and numerous vaccine studies.

DNA↗
PubMed2026

Molecular epidemiology and antimicrobial resistance of human Streptococcus suis isolates in Guangxi, China, 2015-2021.

BACKGROUND: Streptococcus suis (S. suis) is an important zoonotic pathogen and a common colonizer of the upper respiratory tract of pigs. Human infections have been reported in several regions of China, including Guangxi, but genomic and antimicrobial resistance data from this region remain limited. This study investigated the molecular epidemiology, antimicrobial susceptibility, and genomic characteristics of human S. suis isolates collected in Baise City, Guangxi, from 2015 to 2021. METHODS: This retrospective study included 39 non-duplicate clinical isolates confirmed as S. suis by whole-genome analysis. Antimicrobial susceptibility testing was performed using a broth microdilution-based system and interpreted according to the Clinical and Laboratory Standards Institute guidelines. Serotypes were determined by agglutination using type-specific antisera. Whole-genome sequencing was used for species confirmation, multilocus sequence typing, detection of antimicrobial resistance and virulence-associated genes, and core-protein phylogenetic analysis. RESULTS: The median patient age was 55 years, and 36/39 (92.3%) patients were male. Meningitis was documented in 34/39 (87.2%) patients, and hearing impairment occurred in 21/39 (53.8%). Pig- or pork-related exposure was recorded in 15/39 (38.5%) patients. Resistance was highest to tetracycline (38/39, 97.4%), followed by erythromycin and clindamycin (26/39, 66.7% each). Four isolates (10.3%) showed intermediate susceptibility to penicillin, but none were resistant. All isolates remained susceptible to ampicillin, ceftriaxone, levofloxacin, linezolid, vancomycin, and meropenem. Serotype 2 predominated (32/39, 82.1%), followed by serotype 14 (7/39, 17.9%), while ST1 (29/39, 74.4%) and ST7 (7/39, 17.9%) were the two major sequence types. Resistance genes were mainly associated with tetracyclines, macrolides, lincosamides, and aminoglycosides. All ST1 isolates carried mrp and lacked tet(40), while all ST7 isolates showed the reverse pattern. CONCLUSION: Serotype 2 and ST1 predominated among the human S. suis isolates collected at this center. Resistance to tetracycline, erythromycin, and clindamycin was common, while susceptibility to the β-lactams tested was largely preserved. Differences in virulence- and resistance-associated gene profiles were also observed between the major lineages, indicating distinct genetic characteristics among the locally circulating isolates.

Streptococcus suis↗
PubMed2006

A novel HLA-B allele, B*5612, identified by sequence-based typing method.

HLA-B*5612, found in Taiwan using sequence-based typing method, was identical to HLA-B*5502 in exon 2 but differed in exon 3 by 10 nucleotide substitutions at positions 353-420 leading to five amino acid change at codon 94, 95, 97, 103 and 116. As this sequence motif was not found in the Asian population, it is likely that HLA-B*5612 is the product of a complex mechanism of implying a dual gene conversion event.

Base Sequence↗
PubMed1992

Molecular identification of Wolbachia, the agent of cytoplasmic incompatibility in Drosophila simulans, and variability in relation with host mitochondrial types.

Sequences of a segment of the 16S ribosomal DNA of Wolbachia, a rickettsia-like microorganism responsible for cytoplasmic incompatibility in Drosophila simulans, have been obtained after polymerase chain reaction (PCR) amplification. Their comparison with other eubacterial sequences allows us to assign these endosymbionts to the alpha subdivision of purple bacteria. Four related sequences have been obtained for microorganisms carried by eight isofemale lines representative of the three mitochondrial types of D. simulans. Their phylogeny and level of divergence do not parallel that of the mitochondrial DNA, suggesting that several independent infections occurred. There is no direct relation between bacterial phylogeny and formerly identified incompatibility types.

Animals↗
PubMed2005

Using multilocus sequence data to define the pneumococcus.

We investigated the genetic relationships between serotypeable pneumococci and nonserotypeable presumptive pneumococci using multilocus sequence typing (MLST) and partial sequencing of the pneumolysin gene (ply). Among 121 nonserotypeable presumptive pneumococci from Finland, we identified isolates of three classes: those with sequence types (STs) identical to those of serotypeable pneumococci, suggesting authentic pneumococci in which capsular expression had been downregulated or lost; isolates that clustered among serotypeable pneumococci on a tree based on the concatenated sequences of the MLST loci but which had STs that differed from those of serotypeable pneumococci in the MLST database; and a more diverse collection of isolates that did not cluster with serotypeable pneumococci. The latter isolates typically had sequences at all seven MLST loci that were 5 to 10% divergent from those of authentic pneumococci and also had distinct and divergent ply alleles. These isolates are proposed to be distinct from pneumococci but cannot be resolved from them by optochin susceptibility, bile solubility, or the presence of the ply gene. Complete resolution of pneumococci from the related but distinct population is problematic, as recombination between them was evident, and a few isolates of each population possessed alleles at one or occasionally more MLST loci from the other population. However, a tree based on the concatenated sequences of the MLST loci in most cases unambiguously distinguished whether a nonserotypeable isolate was or was not a pneumococcus, and the sequence of the ply gene fragment was found to be useful to resolve difficult cases.

Chromosome Mapping↗
PubMed2006

Molecular characterization of methicillin-resistant Staphylococcus aureus spread by neonates transferred from primary obstetrics clinics to a tertiary care hospital in Korea.

OBJECTIVE: To investigate the characteristics and origins of methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from neonatal patients admitted to a tertiary care hospital from local and primary care obstetrics clinics. DESIGN: Molecular typing study. SETTING: A 1,278-bed tertiary care hospital (Samsung Medical Center) and 2 primary obstetrics clinics in Seoul, Korea. PATIENTS: The genotypic characteristics of 12 MRSA samples isolated from 11 neonatal patients transferred from 2 primary care obstetrics clinics to a tertiary care hospital were investigated by means of multilocus sequence typing, spa (staphylococcal protein A) typing, and SCCmec typing. Ten MRSA strains isolated from workers and environments in the associated obstetrics clinics were also investigated. RESULTS: Although the antibiograms of isolates from 2 obstetrics clinics differed, no strain showed multidrug resistance to antimicrobials. Multilocus sequence typing analysis showed that all 22 MRSA isolates analyzed in this study had sequence type 1 (with the allelic profile 1-1-1-1-1-1-1), sequence type 493 (62-1-1-1-1-1-1), or a novel sequence type (25-1-1-1-1-1-1) and that all belonged to a single clonal complex (clonal complex 1). Moreover, they all contained SCCmec type IVA and the identical spa type (UJEBKBP). These genotypic characteristics are similar to those of typical community-associated MRSA strains rather than the hospital-acquired MRSA strains common in Korea. CONCLUSION: The findings of this study suggest that community-acquired MRSA strains can spread in primary care clinics and be imported into tertiary care settings.

Bacterial Typing Techniques↗
PubMed2006

A simple model to explain three-base periodicity in coding DNA.

A simple model is put forward to explain the long-known three-base periodicity in coding DNA. We propose the concept of same-phase triplet clustering, i.e. a condition wherein a triplet appears several times in one phase without interruption by the two other possible phases. For instance, in the sequence (i): NTT_GNN_NTT_GNN_NTT_GNN_NNN_NTT_GNN (where N is any nucleotide but combinations producing TTG are excluded) there would be clustering of same-phase TTG because this triplet appears uninterruptedly in phase 2. In contrast, in the sequence (ii): TTG_NTT_GNN_NNT_TGN_NNN_NTT_GNN there is no same-phase clustering because neighboring TTGs are all in different phases. Observe also that in sequence (i) TTG triplets are separated by 3, 3 and 6 nucleotides (3n distances), while in sequence (ii) they are separated by 1, 4 and 5 nucleotides (non-3n distances). In this work, we demonstrate that in coding DNA the 3n distances generated by (i)-type sequences proportionally outnumber the non-3n distances generated by (ii)-type sequences, this condition would be the basis of three-base periodicity. Randomized sequences had (i)- and (ii)-type sequences too but clustering was statistically different. To prove our model we generated (i)-type sequences in a randomized sequence by inducing clustering of same-phase triplets. In agreement with the model this sequence displayed three-base periodicity. Furthermore, two- and four-base periodicities could also be induced by artificially inducing clustering of duplets and tetraplets.

DNA↗
PubMed2001

Previously unknown and phylogenetically diverse members of the green nonsulfur bacteria are indigenous to freshwater lakes.

The phylogenetic diversity of green nonsulfur bacteria in nine stratified freshwater lakes was investigated. A set of oligonucleotide primers was developed that permitted the selective amplification of 16S rRNA gene sequences of this group. Subsequently, amplification products were separated by denaturing gradient gel electrophoresis (DGGE) and sequenced, which yielded a total of 19 novel sequence types. Ten of the sequences were related to those of different cultivated members of the C hloroflexus assemblage, whereas nine fell into the T78 group of environmental clones. For the latter subgroup of the green nonsulfur bacteria, no molecular isolate from freshwater plankton has been reported so far. Several of the sequence types occurred in more than one lake, indicating that not only relatives of the C hloroflexus assemblage, but also bacteria of the clone T78 group represent indigenous bacteria of nonthermal stratified freshwater ecosystems. Our results indicate that the natural diversity in the phylum of the green nonsulfur bacteria has been significantly underestimated in the past.

Bacteria↗
PubMed2025

Molecular epidemiology of panton valentine leukocidin-producing Staphylococcus aureus infections, Djibouti, 2018-2023.

Between 2018 and 2023, a genomic study was conducted in a military camp in Djibouti to investigate the molecular epidemiology of Panton-Valentine Leukocidin-producing Staphylococcus aureus. Among 43 isolates, Sequence Type 152 was predominant (72%), mainly associated with spa types t355 and t4235. Core-genome Multi-Locus Sequence Typing revealed two concurrent transmission dynamics: localized inter-human outbreaks and repeated introductions from external sources. Comparative genomics with other African Sequence Type 152 isolates showed similar levels of diversity, suggesting a widespread continental dissemination. These findings highlight the importance of genomic surveillance to better understand and control the spread of this virulent lineage in Africa.

Leukocidins↗
PubMedDate not supplied

Holistic versus composite preferences for lifetime treatment sequences for type 2 diabetes.

This study of patient preferences for lifetime treatment sequences for type 2 diabetes had three objectives: to assess the feasibility of obtaining holistic preference assessments using the time-tradeoff (TTO) technique; to compare composite and holistic preference scores for the same lifetime treatment paths; and to assess the validity of composite and holistic preference measures in terms of their congruence with an individual's rank-order preferences. 101 persons with type 2 diabetes provided preference ratings for hyperglycemic treatments lasting 30 years, including eight discrete treatment states and four treatment paths. Scenarios described drug and glucose-testing regimens, efficacy of glucose control, and side effects. After ranking and rating scenarios on a thermometer scale, subjects provided TTO preferences for each treatment state or path scenario. Holistic assessment of treatment paths was feasible with the TTO technique, in terms of useable data (88% of interviews) and effect on coefficients of variation. Holistic and composite preference scores were not statistically different. Agreement was poor between rankings implied by holistic and composite scores and direct rankings. The authors conclude that lifetime treatment paths with minor differences in health effects can be assessed using either composite (QALY) or holistic (HYE) measures. The validity of these TTO-based preference measures remains unknown.

Adult↗
PubMed2002

Evolutionary relationship of Alw26I, Eco31I and Esp3I, restriction endonucleases that recognise overlapping sequences.

Type II restriction endonucleases (ENases) have served as models for understanding the enzyme-based site-specific cleavage of DNA. Using the knowledge gained from the available crystal structures, a number of attempts have been made to alter the specificity of ENases by mutagenesis. The negative results of these experiments argue that the three-dimensional structure of DNA-ENase complexes does not provide enough information to enable us to understand the interactions between DNA and ENases in detail. This conclusion calls for alternative approaches to the study of structure-function relationships related to the specificity of ENases. Comparative analysis of ENases that manifest divergent substrate specificities, but at the same time are evolutionarily related to each other, may be helpful in this respect. The success of such studies depends to a great extent on the availability of related ENases that recognise partially overlapping nucleotide sequences (e.g. sets of enzymes that bind to recognition sites of increasing length). In this study we report the cloning and sequence analysis of genes for three Type IIS restriction-modification (RM) systems. The genes encoding the ENases Alw26I, Eco31I and Esp3I (whose recognition sequences are 5'-GTCTC-3', 5'-GGTCTC-3' and 5'-CGTCTC-3', respectively) and their accompanying methyltransferases (MTases) have been cloned and the deduced amino acid sequences of their products have been compared. In pairwise comparisons, the degree of sequence identity between Alw26I, Eco31I and Esp3I ENases is higher than that observed hitherto among ENases that recognise partially overlapping nucleotide sequences. The sequences of Alw26I, Eco31I and Esp3I also reveal identical mosaic patterns of sequence conservation, which supports the idea that they are evolutionarily related and suggests that they should show a high level of structural similarity. Thus these ENases represent very attractive models for the study of the molecular basis of variation in the specific recognition of DNA targets. The corresponding MTases are represented by proteins of unusual structural and functional organisation. Both M. Alw26I and M. Esp3I are represented by a single bifunctional protein, which is composed of an m(6)A-MTase domain fused to a m(5)C-MTase domain. In contrast, two separate genes encode the m(6)A-MTase and m(5)C-MTase in the Eco31I RM system. Among the known bacterial m(5)C-MTases, the m(5)C-MTases of M. Alw26I, M. Eco31I and M. Esp3I represent unique examples of the circular permutation of their putative target recognition domains together with the conserved motifs IX and X.

Acinetobacter↗
PubMed2000

Discrimination between clinically relevant and nonrelevant Acanthamoeba strains isolated from contact lens- wearing keratitis patients in Austria.

Eighteen cases of Acanthamoeba-associated keratitis among contact lens wearers seen at the Department of Ophthalmology, Karl-Franzens-University, Graz, Austria, between 1996 and 1999 are reviewed. The amoebae were proven to be the causative agents in three patients. The aim of our study was to discriminate between clinically relevant and nonrelevant isolates and to assess the relatedness of the isolates to published strains. Altogether, 20 strains of free-living amoebae, including 15 Acanthamoeba strains, 3 Vahlkampfia strains, and 2 Hartmannella strains, were isolated from clinical specimens. The virulent Acanthamoeba strains were identified as A. polyphaga and two strains of A. hatchetti. To our knowledge this is the first determination of keratitis-causing Acanthamoeba strains in Austria. Clinically relevant isolates differed markedly from nonrelevant isolates with respect to their physiological properties. 18S ribosomal DNA sequence types were determined for the three physiologically most-divergent strains including one of the keratitis-causing strains. This highly virulent strain exhibited sequence type T6, a sequence type not previously associated with keratitis. Sequence data indicate that Acanthamoeba strains causing keratitis as well as nonpathogenic strains of Acanthamoeba in Austria are most closely related to published strains from other parts of the world. Moreover, the results of our study support the assumption that pathogenicity in Acanthamoeba is a distinct capability of certain strains and not dependent on appropriate conditions for the establishment of an infection.

Acanthamoeba↗
PubMed2004

Use of highly variable intergenic spacer sequences for multispacer typing of Rickettsia conorii strains.

By use of the nearly perfectly colinear genomes of Rickettsia conorii and Rickettsia prowazekii, we compared the usefulness of three types of sequences for typing of R. conorii isolates: (i) 5 variable coding genes comprising the 16S ribosomal DNA, gltA, ompB, and sca4 (gene D) genes, which are present in both genomes, and the ompA gene, which is degraded in R. prowazekii; (ii) 28 genes degraded in R. conorii but intact in R. prowazekii, including 23 split and 5 remnant genes; and (iii) 27 conserved and 25 variable intergenic spacers. The 4 conserved and 23 split genes as well as the 27 conserved intergenic spacers each had identical sequences in 34 human and 5 tick isolates of R. conorii. Analysis of the ompA sequences identified three genotypes of R. conorii. The variable intergenic spacers were significantly more variable than conserved genes, split genes, remnant genes, and conserved spacers (P < 10(-2) in all cases). Four of the variable intergenic spacers (dksA-xerC, mppA-purC, rpmE-tRNA(fMet), and tRNA(Gly)-tRNA(Tyr)) had highly variable sequences; when they were combined for typing, multispacer typing (MST) identified 27 different genotypes in the 39 R. conorii isolates. Two batches from the same R. conorii strain, Malish (Seven), with different culture passage histories were found to exhibit the same MST type. MST was more discriminatory for strain genotyping than multiple gene sequencing (P < 10(-2)). Phylogenetic analysis based on MST sequences was concordant with the geographic origins of R. conorii isolates. Our study supports the usefulness of MST for strain genotyping. This tool may be useful for tracing a strain and identifying its source during outbreaks, including those resulting from bioterrorism.

Animals↗
PubMed1994

MRI of orofacial tumors and paragangliomas with 2D GE sequences: indications and optimal sequence parameters.

The aim of this paper is to determine to what extent and in which cases 2D gradient echo (2D GE) sequences can be applied alternatively or additionally to spin echo (SE) sequences for improved diagnostic evaluation. Imaging with SE sequences is the most frequently used MR technique in the assessment of ear, nose and throat (ENT) tumors. In literature there are only a few reports on the contrast behaviour of 2D GE sequences using different sequence parameters and their application in ENT tumors. This paper set out to establish the most suitable sequence type and sequence parameters. Measurements were performed with a Magnetom SP 63 MR system (Siemens) with a field strength of 1.5 T, using head and Helmholtz coils. One-hundred twenty-eight volunteers and 369 patients were examined with 2D GE sequences. In order to find the best MR technique for the examination of orofacial tumors and paragangliomas, FLASH, FISP and PSIF sequences with different sequence parameters (TR, TE, flip angle, bandwidth) were applied. The results of these examinations confirmed the superiority of 2D GE sequences over SE sequences. In conclusion, contrast enhanced or unenhanced T1 weighted SE sequences should be replaced by 2D GE FLASH 70 degrees in the examination of orofacial tumors. In the case of paragangliomas of the jugular bulb and the parapharyngeal space T1- and T2-weighted SE sequences should be replaced by 2D GE FLASH 40 degrees.

Head and Neck Neoplasms↗