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PubMed1995

Predictive value of precore hepatitis B virus mutations in spontaneous and interferon-induced hepatitis B e antigen clearance.

We previously reported two mutually exclusive mutations in the precore region of hepatitis B virus: M1 (T-1856, proline-serine substitution at codon 15) and M2 (A-1896, stop codon at codon 28). This study was conducted to determine if the presence of precore mutants affect spontaneous or interferon (IFN)-induced hepatitis B e antigen (HBeAg) clearance. Sera from 201 hepatitis B e antigen positive Chinese patients (including 106 who participated in a controlled trial of IFN therapy) with chronic hepatitis B virus (HBV) infection were analyzed by direct sequencing of HBV DNA after amplification by polymerase chain reaction (PCR) assay. Forty-three (21%) patients had M1 (T-1856), and 20 patients (10%) had M2 (A-1896). During a follow-up period of 1 to 7 years, 75%, 28%, and 26% of those with M2 (A-1896), M1 (T-1856), and wild type sequence respectively, cleared HBeAg (P < .0001). Eighteen (67%) of 27 patients with wild-type sequence but none of 10 patients who had M1 (T-1856) in their initial samples developed M2 (A-1896) after loss of HBeAg (P < .0001). Sustained antiviral response was achieved in 55%, 0%, and 17% of interferon-treated patients who had M2 (A-1896), M1 (T-1856), and wild-type sequence, respectively, initially (P = .04). However, patients with M2 (A-1896) were also more likely to have elevated pretreatment aminotransferase levels (P = .02). In summary, HBeAg-positive Chinese patients with M2 (A-1896) were mor likely to clear HBeAg, and to do so earlier.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed2004

Allelic diversity and population structure in Oenococcus oeni as determined from sequence analysis of housekeeping genes.

Oenococcus oeni is the organism of choice for promoting malolactic fermentation in wine. The population biology of O. oeni is poorly understood and remains unclear. For a better understanding of the mode of genetic variation within this species, we investigated by using multilocus sequence typing (MLST) with the gyrB, pgm, ddl, recP, and mleA genes the genetic diversity and genetic relationships among 18 O. oeni strains isolated in various years from wines of the United States, France, Germany, Spain, and Italy. These strains have also been characterized by ribotyping and restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 16S-23S rRNA gene intergenic spacer region (ISR). Ribotyping grouped the strains into two groups; however, the RFLP analysis of the ISRs showed no differences in the strains analyzed. In contrast, MLST in oenococci had a good discriminatory ability, and we have found a higher genetic diversity than indicated by ribotyping analysis. All sequence types were represented by a single strain, and all the strains could be distinguished from each other because they had unique combinations of alleles. Strains assumed to be identical showed the same sequence type. Phylogenetic analyses indicated a panmictic population structure in O. oeni. Sequences were analyzed for evidence of recombination by split decomposition analysis and analysis of clustered polymorphisms. All results indicated that recombination plays a major role in creating the genetic heterogeneity of O. oeni. A low standardized index of association value indicated that the O. oeni genes analyzed are close to linkage equilibrium. This study constitutes the first step in the development of an MLST method for O. oeni and the first example of the application of MLST to a nonpathogenic food production bacteria.

Alleles↗
PubMed1999

Oropharyngeal samples for genotyping and monitoring response to treatment in AIDS patients with Pneumocystis carinii pneumonia.

A nested PCR, amplifying a portion of the gene encoding the mitochondrial large subunit ribosomal RNA (mt LSU rRNA) of Pneumocystis carinii sp. f. hominis was applied to oropharyngeal samples obtained on repeated occasions from 12 HIV-infected patients with P. carinii pneumonia (PCP) to monitor response to anti-P. carinii treatment. Genotyping of P. carinii sp. f. hominis was also performed on paired samples of oropharyngeal and broncho-alveolar lavage samples before the start of treatment, and on oropharyngeal samples during the course of treatment, by analysis of sequence variation at the internal transcribed spacer (ITS) regions of the nuclear rRNA operon. When a simple dilutional method was used, a reduction in the amount of amplification product was observed in samples from all patients during the course of treatment. In eight of the 12 patients, a single ITS sequence type was found in the oropharyngeal samples and also in the paired broncho-alveolar lavage sample. A mixed infection was identified in the samples from three patients. In eight patients, the ITS sequence types identified in the oropharyngeal sample were the same as in the broncho-alveolar lavage sample. Nested PCR amplifying the mt LSU rRNA on oropharyngeal samples provides a non-invasive method of monitoring response to treatment of PCP. ITS sequence typing of P. carinii sp. f. hominis from oropharyngeal samples appears to be a reliable alternative to broncho-alveolar lavage samples and provides a non-invasive tool for further epidemiological studies.

AIDS-Related Opportunistic Infections↗
PubMed2006

Dramatic increase in a single genotype of TRNG ciprofloxacin-resistant Neisseria gonorrhoeae isolates in men who have sex with men.

In 2003, episodes of gonorrhoea caused by ciprofloxacin-resistant strains increased to 15.3% from 11% in 2002. This was coincident with a marked increase in strains characterized as serogroup WI, ciprofloxacin-resistant bearing the tetracycline resistance plasmid. Molecular typing of these strains, using Neisseria gonorrhoeae multiantigen sequence typing (NG-MAST) revealed 71% (34/48) were of the same sequence type, ST338, accounting for 4.1% (34/824) of all strains in 2003. Epidemiological data demonstrated that transmission of ST338 was associated with men who have sex with men (MSM; 23/27), acquisition within the UK (22/26) and having two or more partners in the previous three-month period (18/27). The combined use of highly discriminatory typing and epidemiological surveillance helps to identify successful transmission networks.

Anti-Infective Agents↗
PubMed1999

Sequence polymorphism in the mtDNA HV1 region in Japanese and Chinese.

We investigated the nucleotide substitution and insertion/deletion polymorphism of the HV1 region in mtDNA by sequencing blood samples from 150 unrelated Japanese and 120 unrelated Chinese and revealed 108 sequence types from the Japanese group and 87 sequence types from the Chinese. Some substitutions were characteristic of East Asian populations as compared with data reported on Caucasian populations, and some were area-specific among East Asians. The level of genetic diversity and genetic identity revealed by this system was superior to that obtained by VNTR systems for nuclear DNA. These results show the usefulness of mtDNA sequencing in forensic examination for individual identification. We also found some sequence variations in the homopolymeric tract of cytosine (np16180-16194 in the Anderson's reference sequence) that might suggest some hints regarding the mechanisms for and the development of heteroplasmic length variations in this tract.

Journal Article↗
PubMed2005

Use of molecular diversity of Mycoplasma gallisepticum by gene-targeted sequencing (GTS) and random amplified polymorphic DNA (RAPD) analysis for epidemiological studies.

A total of 67 Mycoplasma gallisepticum field isolates from the USA, Israel and Australia, and 10 reference strains, were characterized by gene-targeted sequencing (GTS) analysis of portions of the putative cytadhesin pvpA gene, the cytadhesin gapA gene, the cytadhesin mgc2 gene, and an uncharacterized hypothetical surface lipoprotein-encoding gene designated genome coding DNA sequence (CDS) MGA_0319. The regions of the surface-protein-encoding genes targeted in this analysis were found to be stable within a strain, after sequencing different in vitro passages of M. gallisepticum reference strains. Gene sequences were first analysed on the basis of gene size polymorphism. The pvpA and mgc2 genes are characterized by the presence of different nucleotide insertions/deletions. However, differentiation of isolates based solely on pvpA/mgc2 PCR size polymorphism was not found to be a reliable method to differentiate among M. gallisepticum isolates. On the other hand, GTS analysis based on the nucleotide sequence identities of individual and multiple genes correlated with epidemiologically linked isolates and with random amplified polymorphic DNA (RAPD) analysis. GTS analysis of individual genes, gapA, MGA_0319, mgc2 and pvpA, identified 17, 16, 20 and 22 sequence types, respectively. GTS analysis using multiple gene sequences mgc2/pvpa and gapA/MGA_0319/mgc2/pvpA identified 38 and 40 sequence types, respectively. GTS of multiple surface-protein-encoding genes showed better discriminatory power than RAPD analysis, which identified 36 pattern types from the same panel of M. gallisepticum strains. These results are believed to provide the first evidence that typing of M. gallisepticum isolates by GTS analysis of surface-protein genes is a sensitive and reproducible typing method and will allow rapid global comparisons between laboratories.

Adhesins, Bacterial↗
PubMed1986

Complete nucleotide sequence of type 6 M protein of the group A Streptococcus. Repetitive structure and membrane anchor.

The DNA sequence of the gene for type 6 M protein of Streptococcus pyogenes contains two extended tandem repeat regions and one nontandem repeat region. We suggest that the duplication and deletion of these repeats generates the observed diversity in size and sequence among the family of M proteins in the group A streptococci. In addition, the DNA sequence reveals the presence of a 42-amino-acid signal peptide, a region rich in proline that is thought to be located in the cell wall, and a membrane anchor sequence at the carboxyl-terminal end of the protein. Signals similar to the consensus sequences recognized for the initiation of transcription and translation in Gram-positive bacteria have been identified in the DNA sequence. Codon usage is similar to that of other Gram-positive bacteria and significantly different from that of Escherichia coli.

Amino Acid Sequence↗
PubMed2005

Nucleotide polymorphism in the Adh2 region of the wild rice Oryza rufipogon.

DNA variation in the alcohol dehydrogenase (Adh2) region of the wild rice Oryza rufipogon and its related species was analyzed to clarify maintenance mechanisms of the DNA variation in these species. A dimorphic pattern was detected in the Adh2 region of O. rufipogon. The silent nucleotide diversity (pi) in the Adh2 region in O. rufipogon was 0.011, which was higher than that of the Adh1 region in O. rufipogon. Especially, a high nucleotide diversity was detected at synonymous sites of the catalytic domain 1. Average nucleotide diversity at silent sites within each of the dimorphic sequence types of the Adh2 region was similar to that in the Adh1 region, indicating that the high level of silent polymorphism in the Adh2 region was caused by the difference between the dimorphic sequence types. On the other hand, the level of replacement polymorphism in the Adh2 region was as low as that in the Adh1 region. The neutrality test of Fu and Li indicated significantly negative deviation from the neutral mutation model for the replacement sites of the Adh2 region. This result suggests purifying selection on the replacement sites of the Adh2 region, as detected for the Adh1 region. Significant linkage disequilibria (16.4% of the tests) were detected between the Adh1 and Adh2 regions. Even when nonrandom association was tested for the strains belonging to one of the divergent sequence types of the Adh2 region, significant interlocus linkage disequilibria were detected. The close physical distance and/or epistasis between the two Adh regions could be invoked to explain these nonrandom associations.

Alcohol Dehydrogenase↗
PubMed2006

Genotyping of pathogenic Acanthamoebae isolated from clinical samples in Greece--report of a clinical isolate presenting T5 genotype.

Amoebae belonging to the genus Acanthamoeba are potentially pathogenic to humans, causing mainly amoebic keratitis. Pathogenic ability of the 15 known Acanthamoeba genotypes is under investigation. We report that four out of five cases of amoebic keratitis studied in Greece, present T4 sequence type, while the remaining one presents T5 sequence type (Acanthamoeba lenticulata), which is the second most frequent genotype found among environmental samples. Thus, it is confirmed, for the first time to our knowledge, that A. lenticulata can cause keratitis. However the reason that it is under represented in clinical samples compared to environmental ones is unknown.

Acanthamoeba↗
PubMed2006

Identification of novel chimpanzee MHC class I and II alleles using an improved sequence-based typing strategy.

The chimpanzee (Pan troglodytes) is a valuable model for the study of infection with hepatitis C virus and hepatitis B virus, to which only humans and chimpanzees are susceptible. Because the cellular immune response plays a crucial role in host defense against these viruses, the analysis of major histocompatibility complex (MHC) (Patr) class I and II allele diversity in chimpanzees is essential for immune response analysis and vaccine development. In the present study, we report a novel, rapid, and sensitive sequence-based typing strategy to identify polymorphisms of the principal Patr class I (Patr-A and Patr-B) and Patr class II (Patr-DQB1 and Patr-DRB1) alleles. Using this method we identified seven novel Patr alleles in 17 chimpanzees, one of them present in 3 chimpanzees. Furthermore, we detected heterozygosity more rapidly and with higher sensitivity than was done with previous techniques that were based on reverse transcription and amplification of messenger RNA followed by molecular cloning and sequencing.

Alleles↗
PubMed1996

Intragenic recombination in the Adh locus of the wild plant Arabidopsis thaliana.

Nucleotide variation in the Adh region of the wild plant Arabidopsis thaliana was analyzed in 17 ecotypes sampled worldwide to investigate DNA polymorphism in natural plant populations. The investigated 2.4-kb Adh region was divided into four blocks by intragenic recombinations between two parental sequence types that diverged 6.3 million years (Myr) ago, if the nucleotide mutation rate mu = 10(-9) is assumed. Within each block, dimorphism of segregating variations was observed with intermediate frequencies, which caused a substantial amount of nucleotide variation in A. thaliana at the species level. The first recombination introduced the divergent variation that resulted in dimorphism in this plant species approximately 3.3 Myr ago, and three subsequent intragenic combinations have occurred sporadically in approximately 1.1-Myr intervals. It was shown that there was only a limited number (six) of sequence types in this species and that no clear association was observed between sequence type and geographic origin. Taken together, these results suggest that A. thaliana has spread over the world only recently. It can be concluded that recombination played an important role in the evolutionary history of A. thaliana, especially through the generation of DNA polymorphism in the natural populations of this plant species.

Alcohol Dehydrogenase↗
PubMed2005

Clonal analysis of invasive pneumococcal isolates in Scotland and coverage of serotypes by the licensed conjugate polysaccharide pneumococcal vaccine: possible implications for UK vaccine policy.

A 7-valent pneumococcal conjugate vaccine (PCV7) has gained licensure and has proven successful in the USA for preventing pneumococcal disease and reducing the incidence of antibiotic-resistant pneumococcal strains. The ability, therefore, to accurately monitor the likely effect of the introduction of PCV7 vaccine on invasive pneumococcal disease in the UK is essential. Serotyping and multilocus sequence typing was performed on invasive isolates of Streptococcus pneumoniae (n=645) from Scotland during 2003. The information gained from this was used to evaluate serotype coverage by the vaccine and the relationship between serotypes. In the present study, invasive pneumococcal disease in Scotland was caused by 33 different serotypes, consisting of 150 sequence types. Overall, 48.4% of the isolates were of serotypes included in the PCV7. Pneumococci were most frequently associated with sequence types 9, 124, and 162. PCV7 would provide protection in 71.8% of infants under 5 years of age against the serotypes in the vaccine. There was limited evidence of the potential for capsule switch among currently circulating invasive pneumococci. The successful implementation of a suitable vaccination programme should lead to a reduction in invasive pneumococcal disease in the UK as well as a reduction in antibiotic resistance of pneumococcal strains.

Child, Preschool↗
PubMed1998

Clusters of Pneumocystis carinii pneumonia: analysis of person-to-person transmission by genotyping.

Genotyping at the internal transcribed spacer (ITS) regions of the nuclear rRNA operon was performed on isolates of P. carinii sp. f. hominis from three clusters of P. carinii pneumonia among eight patients with haematological malignancies and six with HIV infection. Nine different ITS sequence types of P. carinii sp. f. hominis were identified in the samples from the patients with haematological malignancies, suggesting that this cluster of cases of P. carinii pneumonia was unlikely to have resulted from nosocomial transmission. A common ITS sequence type was observed in two of the patients with haematological malignancies who shared a hospital room, and also in two of the patients with HIV infection who had prolonged close contact on the ward. In contrast, different ITS sequence types were detected in samples from an HIV-infected homosexual couple who shared the same household. These data suggest that person-to-person transmission of P. carinii sp. f. hominis may occur from infected to susceptible immunosuppressed patients with close contact within hospital environments. However direct transmission between patients did not account for the majority of cases within the clusters, suggesting that person-to-person transmission of P. carinii sp. f. hominis infection may be a relatively infrequent event and does not constitute the major route of transmission in man.

AIDS-Related Opportunistic Infections↗
PubMed2026

Prevalence, genomic characterization, and biofilm-forming capacity of extended-spectrum &#x3b2;-lactamase-producing Escherichia coli from faecal samples of broiler chickens in Jinan City, China.

The emergence of extended-spectrum &#x3b2;-lactamase (ESBL)-producing Escherichia coli in food animals threatens both veterinary and human medicine by compromising critically important antimicrobials. This study characterized the prevalence, antimicrobial resistance profiles, molecular epidemiology, and virulence attributes of ESBL-producing E. coli isolated from broiler farms in Jinan City, China. From 600 faecal samples, 537 E. coli isolates were recovered (89.5 % isolation rate), with 71 (13.2 %) identified as ESBL producers. Antimicrobial susceptibility testing revealed markedly more severe resistance among ESBL-producing isolates, with complete ampicillin resistance (100 %) and high-level resistance to sulfamethoxazole/trimethoprim (94.37 %), streptomycin (87.32 %), chloramphenicol (78.87 %), and tetracycline (70.42 %). Resistance to extended-spectrum cephalosporins cefuroxime and ceftriaxone reached 43.66 % and 36.62 %, respectively, while amoxicillin/clavulanic acid susceptibility declined to 61.97 %. Whole-genome sequencing identified blaCTX-M-55 (29.58 %) as the predominant ESBL genotype, followed by blaCTX-M-64 (23.94 %), blaCTX-M-15 (19.71 %), blaCTX-M-14 (12.68 %), and blaCTX-M-65 (9.86 %). The plasmid-mediated colistin resistance gene mcr-1 was detected in 25.35 % of ESBL-producing isolates, indicating a substantial reservoir of last-resort antibiotic resistance determinants, though physical linkage between mcr-1 and ESBL-encoding genes remains undetermined due to short-read sequencing limitations. Multilocus sequence typing revealed ST117 (18.3 %) as the predominant sequence type, associated mainly with serogroup O78 (25.4 %). Virulence gene profiling demonstrated high prevalence of the serum survival gene iss (85.9 %), commonly associated with avian pathogenic E. coli, though avian pathogenicity was not experimentally confirmed. Notably, 90.14 % of ESBL-producing isolates demonstrated biofilm-forming capacity, with 16.90 % classified as strong biofilm producers forming mature, mushroom-shaped microcolonies. These findings demonstrate that broiler chickens in the sampled Jinan farms constitute a significant reservoir of multidrug-resistant, biofilm-forming ESBL-producing E. coli harboring clinically relevant genotypes, including mcr-1. The predominance of ST117, reported in both poultry and human clinical settings, along with extensive co-resistance profiles, underscores the value of integrated surveillance and antimicrobial stewardship in poultry production, though direct zoonotic transmission evidence requires further comparative genomic and epidemiological investigation.

Antimicrobial resistance↗
PubMed2003

Infections by Neisseria meningitidis serogroup X in Spain.

We describe an atypical presentation of bacteremia caused by N. meningitidis serogroup X. By multilocus sequence typing the isolate was characterized to the sequence type 2139, which is not related with the clonal complex recently isolated in Africa. Since 1984, only six cases of serogroup X N. meningitidis infections have been diagnosed in Spain. Nevertheless, after the application of the conjugated vaccine, attention should be paid to the emergence of infections caused by unusual serogroups of N. meningitidis.

Anti-Bacterial Agents↗
PubMed2005

Novel antibiotic-resistant pneumococcal strains recovered from the upper respiratory tracts of HIV-infected adults and their children in Kisumu, Kenya.

In a survey of genetic diversity within penicillin-nonsusceptible pneumococcal isolates in Kenya, we examined 162 upper respiratory isolates from 104 human immunodeficiency virus (HIV)-infected adults and 46 children in a cotrimoxazole prophylaxis study. Antibiotic resistance levels were high; 152 (94.4%) were cotrimoxazole nonsusceptible (134 fully resistant) and 124 (77%) were intermediately penicillin resistant. Isolates nonsusceptible to penicillin and cotrimoxazole (PNCNP) were found among 24 of the 29 serotypes encountered, 15 of which have rarely or never had documented nonsusceptibility to penicillin. These included serotypes 3, 4, 7C, 7F, 10A, 11A, 13, 15A, 15B, 16F, 17F, 19B, 21, 35A, and 35B. Segments of pbp2b genes from 9 PNCNP (serotypes 3, 13, 15A, 16F, 20, and 35A) were typical of resistance-conferring alleles in that they were highly divergent and contained two substitutions thought to be critical for resistance. Similarly, the dhfr genes from 3 PNCNP were divergent and contained a substitution required for cotrimoxazole resistance. Multilocus sequence typing (MLST) of 48 PNCNP revealed 33 sequence types (STs), none of which were previously recorded at http://www.mlst.net. Comparisons with all known STs revealed that 23 of these STs were unrelated to other known STs, whereas 10 STs were highly related to STs from internationally disseminated strains, including 2 of the 26 antibiotic-resistant clones recognized by the Pneumococcal Molecular Epidemiology Network. Based upon differing serotypes expressed by strains of identical or closely similar genotypes, there has been an extensive history of capsular switching within seven genetic clusters represented by these 10 STs and related STs described at http://www.mlst.net.

Adult↗
PubMed2006

Changing molecular epidemiology of methicillin-resistant Staphylococcus aureus bloodstream isolates from a teaching hospital in Northern Taiwan.

A changing molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) bloodstream isolates from a university-affiliated hospital in Taiwan during a 4-year interval was demonstrated. The changing epidemiology is due to the introduction of a new epidemic clone (sequence type 5) and a local community clone (sequence type 59) of MRSA into the hospital.

Bacteremia↗
PubMed2000

Simultaneous detection and differentiation of Escherichia coli populations from environmental freshwaters by means of sequence variations in a fragment of the beta-D-glucuronidase gene.

A PCR-based denaturing-gradient gel electrophoresis (DGGE) approach was applied to a partial sequence of the beta-D-glucuronidase gene (uidA) for specific detection and differentiation of Escherichia coli populations according to their uidA sequence variations. Detection of sequence variations by PCR-DGGE and by PCR with direct sequencing correlated perfectly. Screening of 50 E. coli freshwater isolates and reference strains revealed 11 sequence types, showing nine polymorphic sites and an average number of pairwise differences between alleles of the uidA gene fragments (screened fragment length, 126 bp) of 2.3%. Among the analyzed strains a range of dominating to more rarely and/or uniquely observed E. coli sequence types was revealed. PCR-DGGE applied to fecally polluted river water samples simultaneously detected E. coli and generated a fingerprint of the mixed populations by separating the polymorphic uidA amplicons. No significant differences between non-cultivation-based and cultivation-based profiles were observed, suggesting that at least some members of all occurring sequence types could be cultivated. As E. coli is frequently used as a fecal indicator, this work is considered an important step towards a new, practical tool for the differentiation and tracing of fecal pollution in all kinds of waters.

Electrophoresis, Polyacrylamide Gel↗