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PubMed2006

Standardized nonculture techniques recommended for European reference laboratories.

Culture-confirmed diagnosis of meningococcal invasive infections is often hindered by early antibiotic treatment. Nonculture molecular standardized methods are now essential tools for the immediate management of meningococcal infections. The European Monitoring Group on Meningococci (EMGM) recommends the following measures. (1) The implementation of standardized protocols of extraction methods for DNA isolation from clinical specimens for PCR-based identification and genogrouping of Neisseria meningitidis. (2) The use of molecular approaches (sequencing of target genes) for the determination of meningococcal susceptibility to antibiotics, such as sequencing of penA and rpoB genes for susceptibility to penicillin G and rifampicin, respectively. (3) The use of nonculture strain characterization by multilocus sequence typing (MLST) and sequence typing of porA and fetA. These approaches can be implemented either by individual reference laboratories or through collaboration and referral between centres.

Bacterial Outer Membrane Proteins↗
PubMed2006

Antigenic shift and increased incidence of meningococcal disease.

BACKGROUND: The incidence of serogroup C and Y meningococcal disease increased in the United States during the 1990s. The cyclical nature of endemic meningococcal disease remains unexplained. The purpose of this study was to investigate the mechanisms associated with the increase in the incidence of meningococcal disease. METHODS: We characterized an increasing incidence of invasive serogroup C and Y meningococcal disease using population-based surveillance from 1992 through 2001. Isolates were characterized by multilocus sequence typing and antigen sequence typing of 3 outer membrane protein (OMP) genes: porA variable regions (VRs) 1 and 2, porB, and fetA VR. RESULTS: For both serogroups, OMP antigenic shifts were associated with increased incidence of meningococcal disease. For serogroup Y, antigenic shift occurred through amino acid substitutions at all 3 OMPs--PorA VR 1 and 2, PorB, and FetA VR. For serogroup C, antigenic shift involved amino acid substitutions at FetA VR and, in some cases, deletion of the porA gene. On the basis of deduced amino acid sequences, the antigenic changes likely occurred by horizontal gene transfer. CONCLUSIONS: Antigenic shifts were associated with increased incidence of serogroup C and serogroup Y meningococcal disease. For serogroup Y, the changes involved all OMP genes that were studied. Increases in the incidence of meningococcal disease may be caused, in part, by antigenic shift.

Amino Acid Sequence↗
PubMed2006

Molecular typing of meningococci: recommendations for target choice and nomenclature.

The diversity and dynamics of Neisseria meningitidis populations generate a requirement for high resolution, comprehensive, and portable typing schemes for meningococcal disease surveillance. Molecular approaches, specifically DNA amplification and sequencing, are the methods of choice for various reasons, including: their generic nature and portability, comprehensive coverage, and ready implementation to culture negative clinical specimens. The following target genes are recommended: (1) the variable regions of the antigen-encoding genes porA and fetA and, if additional resolution is required, the porB gene for rapid investigation of disease outbreaks and investigating the distribution of antigenic variants; (2) the seven multilocus sequence typing loci-these data are essential for the most effective national, and international management of meningococcal disease, as well as being invaluable in studies of meningococcal population biology and evolution. These targets have been employed extensively in reference laboratories throughout the world and validated protocols have been published. It is further recommended that a modified nomenclature be adopted of the form: serogroup: PorA type: FetA type: sequence type (clonal complex), thus: B: P1.19,15: F5-1: ST-33 (cc32).

Bacterial Outer Membrane Proteins↗
PubMed2006

Genomic backgrounds of drug-resistant Streptococcus pneumoniae in Ankara, Turkey: Identification of emerging new clones.

Streptococcus pneumoniae exhibiting decreased susceptibility to penicillin are isolated with an increasing prevalence in Turkey during the last decade. This study was undertaken to investigate the molecular epidemiology of non-penicillin-susceptible pneumococci isolated in Ankara, Turkey. Among a population of 246 pneumococci, 90 pneumococci with penicillin MIC > or = 0.1 microg/ml were serotyped, genotyped by pulsed-field gel electrophoresis (PFGE), and sequence typed by multilocus sequence typing (MLST). The overall resistance to penicillin, cefotaxime, erythromycin, clindamycin, chloramphenicol, tetracycline, rifampicin, ciprofloxacin, and vancomycin were 36.6%, 4%, 27.6%, 10.9%, 5.3%, 22.4%, 4.5%, 2%, and 0, respectively. The most frequent serotypes were 14, 23B, 9V, 19F, 19A, and 23F. PFGE types represented 17 genetic clusters. PFGE and MLST data revealed that there were isolates identical or closely related to the Spain(9V)-3 ST 156 clone, Portugal(19F)- 21 ST 177 clone, and Spain(23F)-1 ST81 clone. Eleven serotype 14 isolates with emerging resistance to penicillin belonged to the ST 230 complex, a predominantly susceptible clone. Serotype 19A, 19F, and 7F variants of the ST 230 clone were also identified in the study population. Eight serotype 23B isolates with a new ST 1349 (18-13-8-6-3-6-8) created another clone with no relation to the currently defined international clones. Although the pandemic clones Spain(9V)-3, Portugal1(9F)-21, and Spain(23F)-1 are present in our region, the emergence of a new 23B clone with a unique ST and the emergence of resistance in the ST230 clone, has presumably contributed to the increase in the prevalence of drug-resistant pneumococci in Turkey.

Anti-Bacterial Agents↗
PubMed1996

Tetrasomy 18p de novo: identification by FISH with conventional and microdissection probes and analysis of parental origin and formation by short sequence repeat typing.

We report a de novo supernumerary isochromosome 18p in a child with tetrasomy 18p, analyzed by a straightforward combination of cytogenetic and molecular cytogenetic methods. The diagnostic procedure consisted of standard banding techniques and fluorescence in situ hybridization (FISH) with centromere and library DNA probes for chromosome 18, and 18p-specific FISH probes prepared by chromosome dissesction and in vitro amplification. The maternal origin as well as the most probable cell stages of formation of the supernumerary isochromosome were determined by typing of short sequence repeats (SSRs). The pattern of allelic distribution suggests a nondisjunction during meiosis followed by a centromeric misdivision in an early postzygotic mitosis as the most probable mode of isochromosome 18p formation. The combination of the applied methods represents a powerful tool to investigate the nature and the origin of de novo marker chromosomes.

Alleles↗
PubMed2005

HLA-A and HLA-B allele frequencies in a mestizo population from Guadalajara, Mexico, determined by sequence-based typing.

HLA-A and HLA-B genes were typed by DNA sequencing in a mestizo population from Guadalajara, Jalisco, Mexico. Thirty-seven HLA-A and 51 HLA-B alleles were observed in 103 samples. The common typical Amerindian alleles (>5%) and haplotypes (>or=2.0%) found were A*02010101, *24020101, *310102, B*350101, and *4002, and A*310102-B*4002, A*240201-B*350101, and the typical European alleles were A*010101, *29010101, B*1402, B*180101, and A*020101-B*1402, A*020101-B*510101, and A*3002-B*180101. This reflects the blending of the two main parental populations of mestizos: Amerindian and Iberian. Mexicans were found to be relatively closer to the Portuguese than to Spaniards. This proximity may indicate a larger Portuguese influence in Mexicans than previously considered. Present data contribute to the understanding of the genetic structure in Mexico.

Gene Frequency↗
PubMed2000

Identification of a new DRB3*02 allelic variant (DRB3*0209) by high-resolution sequence-based typing.

The HLA-DRB3/B4/B5 sequence-based typing method developed in this study in combination with PCR-SSP, enabled us to identify a new DRB3*02 allele, that was named as DRB3*0209 (GenBank accession number AF148518). This name has been officially assigned by the WHO Nomenclature Committee in May 1999. The new allele differs from DRB3*0207 by one substitution in codon 51 from AGG to ACG and another in codon 60 from TAC to TCC, resulting in aminoacid changes from Arg-->Thr (codon 51) and from Tyr-->Ser (codon 60). The DRB3*0209 allele was discovered in two related North Italian families. The fact that it was present in an hemizygous situation in three members of the paternal family and in one member of the secondary related family enabled us to isolate and sequence the new DRB3 allele without cloning, to identify its association with the DRB1 locus, and to generate an Epstein-Barr virus (EBV)-transformed cell line, now present in our ECBR (European Collection for Biomedical Research) Cell Line Bank.

Alleles↗
PubMed1998

Cyclic nucleotide regulation of type-1 plasminogen activator-inhibitor mRNA stability in rat hepatoma cells. Identification of cis-acting sequences.

Type-1 plasminogen activator-inhibitor (PAI-1) is a major physiologic inhibitor of plasminogen activation. Incubation of HTC rat hepatoma cells with the cyclic nucleotide analogue, 8-bromo-cAMP, causes a dramatic increase in tissue-type plasminogen activator activity secondary to a 90% decrease in PAI-1 mRNA. Although 8-bromo-cAMP causes a modest decrease in PAI-1 transcription, regulation is primarily the result of a 3-fold increase in the rate of PAI-1 mRNA degradation. To determine the cis-acting sequences required for cyclic nucleotide regulation, we have stably transfected HTC cells with chimeric genes containing sequences from the rat PAI-1 cDNA and the mouse beta-globin gene and examined the effect of cyclic nucleotides on the decay rate of these transcripts. The mRNA transcribed from the beta-globin gene is stable and not cyclic nucleotide-regulated, whereas the transcript from a construct containing the beta-globin coding region and the PAI-1 3'-untranslated region (UTR) is destabilized in the presence of 8-bromo-cAMP, suggesting that this response is mediated by sequences in the PAI-1 3'-UTR. Analyses by deletion of sequences from this chimeric construct indicate that, whereas more than one region of the PAI-1 3'-UTR can confer cyclic nucleotide responsiveness, the 3'-most 134-nucleotide sequence alone is sufficient to do so. Insertion of PAI-1 sequences within the beta-globin 3'-UTR confirms that the 3'-most 134 nucleotides of PAI-1 mRNA can confer cyclic nucleotide regulation of stability on a heterologous transcript, suggesting that this sequence may play a major role in hormonal regulation of PAI-1 mRNA stability.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2006

Sequence-based typing reveals a novel DLA-88 allele, DLA-88*04501, in a beagle family.

The dog is an important animal model for solid organ as well as stem cell allo transplantation. Methods such as cellular and serological typing and more recently sequence-based typing (SBT) have been used to discriminate tissue antigen disparity of donor and recipient. We applied SBT for the canine class I (DLA-88) and class II (DLA-DRB1) genes in beagle families prior stem cell transplantation. A novel DLA-88 (DLA-88*04501) allele in combination with a DLA-DRB1*01901 allele was found. Sequence comparison of exons 2 and 3 of the novel allele revealed most sequence identity to the DLA-88*01301 allele (96.15% identity at the nucleotide and 90.65% identity at the protein level).

Alleles↗
PubMed1997

Sequencing of HLA class I genes based on the conserved diversity of the noncoding regions: sequencing-based typing of the HLA-A gene.

We present a sequencing-based typing strategy for the HLA-A locus that is generally applicable to all HLA class I genes. Sequencing-based typing is the method of choice for matching in unrelated bone marrow transplantation on the allelic level. We determined the noncoding sequences of all serological antigens and most of their subtypes and discovered a remarkably conserved diversity characterized by polymorphic sequence motifs. In this study we took advantage of this diversity we uncovered in the 5' flanking region, 5' untranslated region and in the introns 1, 2 and 3, which was related to serological families. We established 12 primer mixes for setting up a PCR-based template preparation. Our strategy is based on the separate amplification of haplotypes and therefore defines the cis/trans linkage of polymorphic sequence motifs. This allowed individual sequencing of the haplotypes in all samples heterozygous for the broad antigens as well as the complete analysis of the polymorphic exons 2 and 3. All templates included the 2nd intron which was used as a priming site for the gene-specific 5' and 3' universal sequencing primers regardless of the amplified haplotypes. The independent sequencing of the haplotypes allows the application of the dye terminator cycle sequencing technique, which is less time-consuming and less-laborious than dye primer chemistry. The lack of heterozygous positions essentially facilitates on the one hand the data analysis and on the other hand the detection of new alleles. Sequencing is only required in one direction due to the absence of peak shift problems. The results will remain unambiguous regardless of a growing HLA sequence data bank since this sequencing technique defines the cis/trans linkage of sequence motifs in more than 95% of the cases.

Base Sequence↗
PubMed2004

Characterization of a new HLA-DRB1*01 allele (HLA-DRB1*010203) in a Caucasian Italian family by using sequence-based typing.

We report the identification of an HLA-DRB1*01 nucleotide sequence variant in three members of a Caucasian Italian family by using sequence-based typing. The nucleotide sequence of exon 2 observed in the new allele is identical to that of HLA-DRB1*010201 except in position 189 (codon 34) where the adenine of the consensus was replaced by a guanine and it was designated officially as HLA-DRB1*010203* by the WHO Nomenclature Committee.

Alleles↗
PubMed2005

Prospective surveillance of community-onset and healthcare-associated methicillin-resistant Staphylococcus aureus isolated from a university-affiliated hospital in Japan.

We conducted a prospective comparative study of community-onset (CO) and healthcare-associated (HA) methicillin-resistant Staphylococcus aureus(MRSA) strains between 2000 and 2001 at Tokyo Women's Medical University Hospital (1,500 beds) in Japan. Of the 172 consecutive MRSA isolates analyzed, 13 (8%) were categorized as CO-MRSA. The mean age of patients with CO-MRSA was significantly younger than that of patients with HA-MRSA. Most CO-MRSA strains were isolated from skin and more likely to be susceptible to erythromycin, clindamycin, tetracycline, levofloxacin, and spectinomycin compared to HA-MRSA isolates. Pulsed-field gel electrophoresis (PFGE) analysis, staphylococcal cassette chromosome mec(SCCmec) typing, and multi-locus sequence typing (MLST) revealed that CO-MRSA strains were divided into the following multi-clones: 3 clone A: II: ST5 (PFGE type: SCCmec type: MLST sequence type); 1 L: II: ST5; 1 H: IV: ST1; 1 I: IV: ST81; 2 D: IV: ST8; 1 B: IV: ST89; 1 B: IV: ST379; and 3 B: IV: ST91. Of the 159 HAMRSA strains, 124 (78%) belonged to a single clone (PFGE clone A: SCCmec type II: tst and sec positive: coagulase type II: multi-drug resistance). Four CO-MRSA strains belonging to PFGE clone B: SCCmec type IV: MLST clonal complex 509 (ST89, 91, 379) had the exfoliative toxin B (etb) genes, but all CO-MRSA and HA-MRSA strains did not possess the Panton-Valentine leukocidin (pvl) genes. These results demonstrate that multiple lineages of CO-MRSA have the potential for dissemination in the community in Japan.

Adolescent↗
PubMed2006

[Endocarditis due to meticillin-resistant Staphylococcus aureus originating from pigs].

A 63-year-old woman with a kidney transplant was admitted with endocarditis caused by meticillin-resistant Staphylococcus aureus (MRSA). Once her antibiotic therapy had been adjusted to the sensitivity-pattern of the bacterial strain she recovered, without the need for surgical intervention. The isolated S. aureus was typed by multi-locus sequence typing as sequence type 398, a MRSA-strain that has recently been isolated from a high percentage of Dutch pigs. This is the first report of a life-threatening infection with this pig MRSA. This strain is genetically different from the globally dispersed nosocomial MRSA-strains, and also from the strains that have been epidemic for several years in the USA as the causative agent ofcommunity-acquired skin infections. The Dutch Working Group on Infection Prevention (WIP) has recently adjusted its guidelines to halt further spread of this strain, and advises that the population at risk (pig breeders, slaughterhouse personnel and veterinarians) be held in isolation when hospitalised until MRSA colonisation has been excluded. The patient described here, however, did not belong to this population at risk.

Animals↗
PubMed2004

A sequencing-based typing method for HLA-DQA1 alleles.

Sequencing-based typing (SBT) is the most comprehensive method for characterizing human leukocyte antigen gene polymorphisms. Development of a SBT method for DQA1 is hampered because of a deletion of codon 56 in nearly half of the known DQA1 alleles. Sequence electropherograms of heterozygous samples comprising a deletion allele and a non-deletion allele display misalignment after codon 56 because of a three base-pair shift in the deletion allele. To overcome this problem, we have designed three group-specific primer sets to selectively amplify the deletion alleles from the nondeletion alleles. DNA samples are initially polymerase chain reaction (PCR)-typed using these primer sets along with an internal positive control primer set specific to growth hormone gene 1 (hGH1). The positive group-specific PCR reactions were selectively repeated without hGH1 control primers, and the amplicons were used as template in sequencing reactions. The sequence data were analyzed to obtain DQA1 types using ABI MatchTools software as well as the newly available Conexio Genomics Assign SBT Genotyping Software. The method was validated using a panel of reference DNA from the University of California, Los Angeles, International DNA Exchange Program. We conclude that the present SBT method is a technically simple and robust procedure to characterize the sequence polymorphisms in exon 2 of DQA1 gene.

Alleles↗
PubMed1991

HLA class II specificities and haplotypes in South Africa detected using polymerase chain reaction and sequence-specific oligonucleotide typing.

DNA sequencing of HLA class II alleles has revealed a degree of polymorphism much greater than was expected on the basis of the standard serological typing methods. Amplification of the polymorphic second exon of the class II genes using the polymerase chain reaction, followed by hybridization with sequence-specific oligonucleotide probes, allows the unambiguous identification of alleles which could not be detected previously. Using the protocols of the Eleventh International Histocompatibility Workshop, we have applied this procedure for the typing of several individuals and their families with suspected alleles that had been observed using serology, cellular typing and restriction fragment length polymorphism (RFLPs). These included an allele related to DRw8 and DRw14, which has only been observed in the mixed ancestral South African population. In addition, unusual combinations of class II genes forming unique haplotypic associations were seen.

Alleles↗
PubMed2004

Ambiguities of human leukocyte antigen-B resolved by sequence-based typing of exons 1, 4, and 5.

The elucidation of the sequences of human leukocyte antigen-B (HLA-B)-exons 1 through 5 has led to an increase of ambiguities with alleles having identical exon 2 and 3 sequences, but differences in other exons. At the moment, 26 HLA-B alleles show such ambiguities which can be resolved by sequencing the exons in which the differences are located. Here we report a sequence-based typing (SBT) strategy for heterozygous sequencing of exons 1, 4, and 5, in addition to the previously described exons 2 and 3. The strategy was validated against a panel of 25 individuals, carrying HLA-B alleles from 33 different allele groups. Correct assignment of all HLA-B alleles was obtained for exons 1 through 5. In addition, the SBT protocol was used to resolve ambiguities in 50 individuals. The ambiguous combinations studied were B*0705/06, B*0801/19N, B*1512/19, B*180101/17N, B*270502/13/0504, B*350101/42/40N, B*390101/0103, B*400102/0101, B*440201/19N/27, and B*510101/11N/0105/30/32. In all cases, sequencing revealed the first allele to be present, except for three individuals with B*07. One of them typed B*0705; the other two were B*0706. The described SBT protocol for sequencing exons 1, 4, and 5 is a valuable tool for resolving ambiguities of HLA-B alleles with differences in these exons, as well as for studying the polymorphism of HLA-B outside exons 2 and 3.

Base Sequence↗
PubMed1999

Identification of the metabolically active members of a bacterial community in a polychlorinated biphenyl-polluted moorland soil.

The presumptive metabolically active members of a bacterial community in a moorland soil in Germany, highly polluted with polychlorinated biphenyls (PCBs), were identified by sequencing of cloned reverse transcription-polymerase chain reaction (RT-PCR) amplification products of 16S rRNA generated from total RNA extracts. Analysis of the 16S rRNA clone library revealed a considerable diversity of metabolically active bacteria in the soil, despite the acidic pH and high concentrations of PCBs. Cloned sequence types clustered within the Proteobacteria (34% alpha-, 33% beta- and 7% gamma-subclasses), the Holophaga-Acidobacterium phylum (14%), the Actinobacteria (6.5%) and the Planctomycetales (2%). Three cloned sequence types were not affiliated to any described phylogenetic group. An unusual feature of this soil was the abundance of sequence types within the beta-subclass of the Proteobacteria, most of which were similar to the 16S rRNA gene sequences of species from only two genera, Burkholderia and Variovorax. Three other numerous 16S rRNA sequence types were similar to the sequences of Sphingomonas species, members of the Rhodopila globiformis group and Acidobacterium capsulatum. Some of the sequence types retrieved were similar to the 16S rRNA sequences of bacterial isolates able to degrade a variety of organic pollutants, including PCBs. As the PCB contamination is the major source of measurable carbon in this soil, some of the 16S rRNA sequence types detected and presumed to represent the metabolically active members of the community indicate the organisms likely to be involved, directly or indirectly, in the utilization of the PCBs as carbon and energy sources.

Bacteria↗
PubMed2005

Listeria monocytogenes in the Chinese food system: strain characterization through partial actA sequencing and tissue-culture pathogenicity assays.

Human listeriosis is generally caused by consumption of ready-to-eat (RTE) foods that are stored for extended periods of time at refrigeration temperatures and that permit the growth of the causative agent, Listeria monocytogenes. Food-consumption patterns in China are undergoing rapid changes and more regular consumption of refrigerated-storage RTE foods may increase the risk of human listeriosis. In total, 40 L. monocytogenes isolates were obtained from food (n=32) and sewage (n=6) samples and from two human listeriosis cases that occurred in China. All isolates were characterized into molecular subtypes by DNA sequencing of the 597 bp 3'-terminal region of the virulence gene actA. Sequence data were used to classify the 40 Chinese L. monocytogenes isolates into sequence types and phylogenetic lineages, and to compare the sequence types of the Chinese isolates with those of isolates from the USA. Phylogenetic analyses showed that the Chinese isolates could be separated into two genetic lineages, with 14 and 26 isolates belonging to lineages I and II, respectively. Lineage II could be subdivided further into two clusters, IIA and IIB. Lineages I and II were identical to the two lineages described previously among US L. monocytogenes isolates. In total, 14 actA sequence types could be differentiated among the 40 Chinese L. monocytogenes isolates; two specific actA sequence types were found among both Chinese and US isolates. Isolates belonging to lineage II showed a significantly lower ability to invade and multiply within human intestinal epithelial Caco-2 cells than lineage I isolates. It was concluded that DNA sequencing of the 3'-terminal region of actA appears to be an effective method for rapid subtype and lineage classification of L. monocytogenes. As strains belonging to lineages I and II have previously been found among isolates from Europe and North America, these results show that L. monocytogenes clonal groups found in China are very similar to those found in the USA. Many L. monocytogenes strains may thus represent globally distributed clonal types. Together with the first description of two human listeriosis cases in China, these data indicate that changes in food-distribution and -consumption patterns in China and other countries will probably lead to the emergence of human listeriosis as a food-safety issue, as virulent strains of this pathogen appear to be present in the Chinese food supply.

Adult↗