Search

Xpert MTB/RIF rapidly detects resistance to rifampicin (RR); however, this test misses I491F-RR conferring the rpoBmutation, common in southern Africa. In addition, Xpert MTB/RIF does not distinguish between viable and dead Mycobacterium tuberculosis(MTB).

ABSTRACTCulturing before DNA extraction represents a major time-consuming step in whole-genome sequencing of slow-growing bacteria, such as Mycobacterium tuberculosis. We report a workflow to extract DNA from frozen isolates without reculturing. Prepared libraries and sequence data were comparable with results from recultured aliquots of the same stocks.

The GRIN3Bgene encodes NR3B, a motoneuron-specific member of the NMDA type of ionotropic glutamate receptors. NR3B reduces the Ca2-permeability as well as the overall current of the receptor response and may thereby protect motoneurons against glutamate-mediated excitotoxicity. We tested whether genetic dysfunction of GRIN3Bis implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS).

ABSTRACTThe findings of recent studies addressing the molecular characteristics of Mycobacterium tuberculosiscomplex isolates have initiated a discussion on the classification of M. africanum, especially of those isolates originating from East Africa (cluster F, subtype II) and displaying phenotypic and biochemical characteristics more similar to those of M. tuberculosis. To further address this question, we analyzed a representative collection of 63 M. tuberculosiscomplex strains comprising 30 M. africanumsubtype I strains, 20 M. africanumsubtype II strains, 10 randomly chosen M. tuberculosisisolates, and type strains of M. tuberculosis, M. bovis, and M. africanumfor the following biochemical and molecular characteristics: single-nucleotide polymorphisms (SNPs) in gyrBand narGHJIand the presence or absence of RD1, RD9, and RD12. For all molecular markers analyzed, subtype II strains were identical to the M. tuberculosisstrains tested. In contrast, the subtype I strains as well as the M. africanumtype strain showed unique combinations of SNPs in gyrBand genomic deletions (the absence of RD9 and the presence of RD12), which proves their independence from M. tuberculosisand M. bovis. Accordingly, all subtype I strains displayed main biochemical characteristics included in the original species description of M. africanum. We conclude that the isolates from West Africa were proved to be M. africanumwith respect to the phenotypic and genetic markers analyzed, while the isolates from East Africa must be regarded as phenotypic variants of M. tuberculosis(genotype Uganda). We propose the addition of the molecular characteristics defined here to the species description of M. africanum, which will allow clearer species differentiation in the future.

In Switzerland lack of exercise is common. To improve physical exercise in both women and men, campaigns should consider the motivation for physical activity with respect to gender. This study explores the patterns of self-reported sport activity, habitual physical activity and physical fitness, and associated indicators of self-reported health, health-relevant attitudes, and resources in an urban Swiss population. Using the Bern Lifestyle Panel questionnaire, 511 women and 413 men, aged 55–65 years, in the German-speaking capital of Switzerland were assessed. From the three surveys carried out from 1996 to 1998, this study used the data from the 1997 survey for a cross-sectional analysis. Women demonstrated a lower prevalence of sport activity than men but a higher prevalence of habitual physical activity. Forty-six percent of the women but 80% of the men reported a high level of fitness. In the logistic regression analysis, sport activity in the women was significantly associated with indicators of health, health-relevant attitudes such as internal and external health locus of control, and social class. Among the men, no association with health-relevant orientation could be found. The probability of habitual physical activity among the women was associated with perceived good health, health-relevant orientation and social class, while related factors among the men were indicators of health. In both women and men, perceived good health was strongly associated with self-reported physical fitness. Additionally, women’s fitness was related to perceived disease-related limitation and indicators of health status. The findings indicate a gender-specific distribution of sport activity, habitual physical activity and fitness. Compared to men, physical activity in women is associated more with health-relevant orientation. Along with indicators of health, attention should be paid to both female and male perception of health and self-reported attitudes when there is an attempt to improve physical activity. Since self-reported health status, disease-related limitations, and habitual physical activity, among others, are strongly affected by sociocultural and structural influences, caution should be kept in drawing general conclusions from the findings.

ABSTRACTThe population structure of 234 Mycobacterium tuberculosiscomplex strains obtained during 1995 and 1997 from tuberculosis patients living in Kampala, Uganda (East Africa), was analyzed by routine laboratory procedures, spoligotyping, and IS6110restriction fragment length polymorphism (RFLP) typing. According to biochemical test results, 157 isolates (67%) were classified as M. africanumsubtype II (resistant to thiophen-2-carboxylic acid hydrazide), 76 isolates (32%) were classified as M. tuberculosis, and 1 isolate was classified as classical M. bovis. Spoligotyping did not lead to clear differentiation of M. tuberculosisand M. africanum, but all M. africanumsubtype II isolates lacked spacers 33 to 36, differentiating them from M. africanumsubtype I. Moreover, spoligotyping was not sufficient for differentiation of isolates on the strain level, since 193 (82%) were grouped into clusters. In contrast, in the IS6110-based dendrogram, M. africanumstrains were clustered into two closely related strain families (Uganda I and II) and clearly separated from the M. tuberculosisisolates. A further characteristic of both M. africanumsubtype II families was the absence of spoligotype spacer 40. All strains of family I also lacked spacer 43. The clustering rate obtained by the combination of spoligotyping and RFLP IS6110analysis was similar for M. africanumand M. tuberculosis, as 46% and 49% of the respective isolates were grouped into clusters. The results presented demonstrate that M. africanumsubtype II isolates from Kampala, Uganda, belong to two closely related genotypes, which may represent unique phylogenetic branches within the M. tuberculosiscomplex. We conclude that M. africanumsubtype II is the main cause of human tuberculosis in Kampala, Uganda.

The population structure of 234 Mycobacterium tuberculosis complex strains obtained during 1995 and 1997 from tuberculosis patients living in Kampala, Uganda (East Africa), was analyzed by routine laboratory procedures, spoligotyping, and IS6110 restriction fragment length polymorphism (RFLP) typing. According to biochemical test results, 157 isolates (67%) were classified as M. africanum subtype II (resistant to thiophen-2-carboxylic acid hydrazide), 76 isolates (32%) were classified as M. tuberculosis, and 1 isolate was classified as classical M. bovis. Spoligotyping did not lead to clear differentiation of M. tuberculosis and M. africanum, but all M. africanum subtype II isolates lacked spacers 33 to 36, differentiating them from M. africanum subtype I. Moreover, spoligotyping was not sufficient for differentiation of isolates on the strain level, since 193 (82%) were grouped into clusters. In contrast, in the IS6110-based dendrogram, M. africanum strains were clustered into two closely related strain families (Uganda I and II) and clearly separated from the M. tuberculosis isolates. A further characteristic of both M. africanum subtype II families was the absence of spoligotype spacer 40. All strains of family I also lacked spacer 43. The clustering rate obtained by the combination of spoligotyping and RFLP IS6110 analysis was similar for M. africanum and M. tuberculosis, as 46% and 49% of the respective isolates were grouped into clusters. The results presented demonstrate that M. africanum subtype II isolates from Kampala, Uganda, belong to two closely related genotypes, which may represent unique phylogenetic branches within the M. tuberculosis complex. We conclude that M. africanum subtype II is the main cause of human tuberculosis in Kampala, Uganda.

The stability of IS6110 restriction fragment length polymorphism (RFLP) patterns of Mycobacterium tuberculosis strains in actual transmission chains has been assessed by analyzing the variability of IS6110 RFLP patterns of strains in fingerprint clusters that have been confirmed by classical epidemiological data. Forty susceptible and 35 drug-resistant (including 17 multidrug-resistant) M. tuberculosis strains obtained from 75 patients living in Germany have been analyzed. The epidemiological relationship among strains within the fingerprint clusters has been verified by family contacts (14 clusters) or by contact tracing of the public health offices (7 clusters). The time spans between the first and the last isolate of one cluster ranged from less than 1 to 29 months. Of the 75 strains only 1 showed a one-band variation when compared to the other nine isolates grouped in the same cluster, corresponding with a rate of change of approximately 1.9% per possible transmission (one index patient per cluster was subtracted from the total number of isolates). These results confirm a high degree of stability of IS6110 RFLP patterns of transmitted M. tuberculosis strains. Furthermore, the data presented indicate that isolates with identical IS6110 DNA fingerprint patterns are a good indicator for the rate of recent transmission in a study population.

Abstract: The enterobacterial repetitive intergenic consensus (ERIC)-PCR method was employed to generate genomic amplification products of Sinorhizobium meliloti strain 2011. Eleven distinctive PCR fragments obtained in PCR reactions by using the ERIC2 primer were cloned and their partial or complete nucleotide sequences established. DNA sequences that extended past the ERIC2 primer region were not conserved among the 11 PCR fragments and showed no sequence similarity to the enterobacterial ERIC consensus sequence. Thus, repetitive ERIC or ERIC-like sequences seem not to be an integral part of the S. meliloti genome. An amplification product of S. meliloti 2011 was identified which was present in S. meliloti strains but absent in other rhizobial species. Based on the nucleotide sequence information, a pair of PCR primers was designed and used for PCR amplification of sequences of S. meliloti laboratory strains 2011, L5–30, AK631 and 102F34. Nucleotide sequence analysis of the amplification products revealed a 100% DNA sequence conservation. Database searches showed that the DNA fragment putatively encodes the C-terminal part of a protein displaying similarity to 2-hydroxyacid dehydrogenases of various organisms. The newly designed PCR primers should be useful for the rapid identification of S. meliloti isolates.

Isothermal P−x data at 453.25 K and excess enthalpies at 363.15 K and 413.15 K are presented for binary systems of benzyl benzoate together with benzene, toluene, phenol, benzaldehyde, and benzyl alcohol. As pointed out previously (Nienhaus, B.; Limbeck, U.; Bölts, R.; de Haan, A. B.; Niemann, S. H.; Gmehling, J. J. Chem. Eng. Data 1998, 43, 941−948), information about the real behavior of systems including these components is very important, especially for the production of phenol by toluene oxidation. In addition, these data will be used for the introduction of a new aromatic ester group (AC−COO−) into modified UNIFAC (Dortmund), which is part of the revision, extension, and further development of this group contribution method. The vapor−liquid equilibrium (VLE) data of four systems were measured using a static apparatus. The corresponding experimental excess enthalpy (H^E) data were determined with the help of an isothermal flow calorimeter. For the simultaneous description of VLE and H^E data the NRTL model was used.

In a comparative study, the PCR-based RAPD and ERIC fingerprint methods were evaluated for their resolving power to discriminate among 21 isolates of a natural Rhizobium meliloti population. PCR fingerprint patterns were analysed by using an automated laser fluorescent (ALF) DNA sequencer, thus allowing the automated on-line storage of data. Results obtained were compared to a classification system using insertion sequence (IS) fingerprinting. Both PCR fingerprint methods were comparable in their ability to resolve differences amongst Rh. meliloti isolates. Grouping of strains on the basis of their RAPD as well as their ERIC fingerprints correlated with grouping of strains according to their IS fingerprints. Moreover, strains displaying identical PCR patterns could be further differentiated according to their IS fingerprints, thus allowing a detailed insight into phylogenetic relationship among strains. The automated evaluation of strain-specific fingerprint patterns has the potential to become a valuable tool for studies of bacterial population genetics. Moreover, the rapid identification of single strains, e.g. pathogens in epidemiological studies seems feasible.

A human genomic DNA fragment, pAM37 (HGM8; D21S22), was mapped to chromosome 21q2.1-q2.21 by in situ hybridization. This segment is therefore situated on the boundary of the “pathological region” of Down syndrome. A genomic restriction map encompassing 35 kb of chromosome 21 was derived and two restriction fragment length polymorphisms (RFLPs) were mapped and characterized. A homologous sequence was detected in the mouse genome but no homologous RNA was detected in a range of human tissues. This DNA segment will contribute to the linkage mapping of chromosome 21 and will facilitate delineation of the pathological region of Down syndrome.

The deliberate release of genetically engineered microorganisms requires a thorough characterization of the microbes in question. For the two bioluminescentRhizobium meliloti strains, L1 and L33 [Selbitschka et al. (1992) Mol Ecol 1: 9–19; Selbitschka et al. (1995) FEMS Microbiol Ecol 16:223–232], designated for field release, the sites of genetic modifications in the chromosomes were sequenced from amplified genomic DNA. This indicated no unexpected alterations in the nucleotide sequence. The bioluminescent phenotype was stably inherited over more than 100 generations in liquid cultures. The presence of the luciferase gene in both strains did not have secondary effects on a variety of metabolic pathways as assessed by the Biolog GN system. A specific polymerase chain reaction amplification, based on the chromosomal insertion site of theluc cassette, allowed the discrimination between the two strains and thus simplifies monitoring. The RecA-deficient strain L1 showed a strongly (more than 90%) reduced ability to nodulate alfalfa in competition with its parent strainR. meliloti 2011 and its RecA+ counterpart L33.

The epidemiological relatedness of drug-resistant Mycobacterium tuberculosis strains isolated in Germany in 1995 was evaluated by the standardized IS6110 fingerprinting method. Altogether, 196 M. tuberculosis isolates from 167 patients were analyzed. A large degree of IS6110 polymorphism was found, ranging from 1 to 20 copies. Multiple isolates from one patient generally remained stable over a period of up to 1 year. However, one strain showed an additional fragment 7 months after the first isolate was obtained. Isolates from 55 patients (33%) showed identical fingerprint patterns or fingerprint patterns that differed only in one band, and thus they were clustered in 22 fingerprint groups. Specific transmission links could be established between members of four groups, e.g., transmission by family contacts. In one case, transmission of a multidrug-resistant strain to a patient initially infected with a drug-susceptible strain could be shown. Besides these fingerprint groups, 30 of the 167 isolates (approximately 18%) could be grouped in two fingerprint clusters with a similarity of at least 78%. Approximately 60% of the patients of these two clusters were known to be immigrants from the former Soviet Union, and one patient is still living in Belarus. In conclusion, our results indicate that (i) transmission of drug-resistant strains contributes substantially to the emergence of drug-resistant tuberculosis in Germany and (ii) drug-resistant M. tuberculosis strains were presumably carried over from the former Soviet Union to Germany by immigrants.

Pregnancy-specific ß1-glycoprotein (PSßG) is a heterogeneous product of the human syncytiotrophoblast, closely related to the CEA-NCA multigene family. In the present study, immunoscreening was carried out with anti-PSßG antibodies to isolate cDNA sequences from a placental ?gt11 expression library. One 1847-bp cDNA clone comprising the major portion of the coding sequence of a putative 48-kd peptide was sequenced and characterized. Hybridization of human genomic DNA to the PSßG sequence revealed a complex pattern of restriction fragments, a finding well in agreement with the assumption that there are several independent PSßG genes. A variable PstI band was found in human DNA. Transfer blot analysis of human placental RNA identified transcripts of 2.2 kb and 1.7 kb that appear transiently with increasing levels of expression during gestation. No hybridization of PSßG cDNA to human RNA from liver, kidney, heart, thyroid, and ovary was observed. In analyses of placental RNA from mouse, goat, sheep, and cow, no correponding transcripts could be detected, and DNA hybridization under low-stringency hybridization conditions resulted in very faint cross-reacting bands, presumably indicating sequences that were scarcely related. However, PSßG-specific DNA sequencies with similar restriction patterns were found in primates. These results are compatible with the assumption of late evolutionary development of certain PSßG sequences.

Abstract: Gnotobiotic systems were used to assess the competitive abilities of bioluminescent Sinorhizobium meliloti strains L1 (RecA−) and L33 (RecA+) for growth and host plant nodulation in the presence of a reconstructed S. meliloti population. Three wild-type strains belonging to infective subgroups of a natural S. meliloti population were chosen as competitors in microcosm studies. Whereas the RecA+ strain L33 dominated the reconstructed population with respect to growth and alfalfa nodulation, the competitiveness of the RecA− strain L1 was reduced compared to that of one of the field strains, but comparable to that of the other field isolates. This result indicates that strain L1, despite its recA mutation, has the potential to compete successfully with a resident S. meliloti population after environmental release.

Isothermal P, x data at 413.65 K, excess enthalpies at 323.15, 363.15, and 413.15 K, and azeotropic data are presented for binary systems containing benzaldehyde, benzene, toluene, and phenol. Reliable information about the real behavior of systems including these components is very important for various chemical processes, e.g., for the production of phenol by toluene oxidation. Furthermore, these data will be used for the revision, extension, and further development of the group contribution method modified UNIFAC (Dortmund). The vapor−liquid equilibrium (VLE) data of four systems were measured using a static apparatus. In the case of the system benzaldehyde + phenol, azeotropic data were determined by distillation. For all systems, the corresponding experimental excess enthalpy (H^E) data were determined with the help of an isothermal flow calorimeter. For the simultaneous description of VLE and H^E data, the NRTL model was used.

Abstract: The impact of biocontrol strain Pseudomonas fluorescens CHA0 and of its genetically modified, antibiotic-overproducing derivative CHA0/pME3424 on a reconstructed population of the plant-beneficial Sinorhizobium meliloti bacteria was assessed in gnotobiotic systems. In sterile soil, the final density of the reconstructed S. meliloti population decreased by more than one order of magnitude in the presence of either of the Pseudomonas strains when compared to a control without addition of P. fluorescens. Moreover, there was a change in the proportion of each individual S. meliloti strain within the population. Plant tests also revealed changes in the nodulating S. meliloti population in the presence of strains CHA0 or CHA0/pME3424. In both treatments one S. meliloti strain, f43, was significantly reduced in its root nodule occupancy. Analysis of alfalfa yields showed a slight but statistically significant increase in shoot dry weight when strain CHA0 was added to the reconstructed S. meliloti population whereas no such effect was observed with CHA0/pME3424.

The role of mutations in genes associated with phenotypic resistance to bedaquiline (BDQ) and delamanid (DLM) in Mycobacterium tuberculosiscomplex (MTBc) strains is poorly characterized. A clear understanding of the genetic variants’ role is crucial to guide the development of molecular-based drug susceptibility testing (DST). In this work, we analyzed all mutations in candidate genomic regions associated with BDQ- and DLM-resistant phenotypes using a whole-genome sequencing (WGS) data set from a collection of 4,795 MTBc clinical isolates from six countries with a high burden of tuberculosis (TB).

We have generated previously transgenic rats that overexpress peripheral myelin protein 22 (PMP22) in Schwann cells. In the nerves of these animals, Schwann cells have segregated with axons to the normal 1:1 ratio but remain arrested at the promyelinating stage, apparently unable to elaborate myelin sheaths. We have examined gene expression of these dysmyelinating Schwann cells using semiquantitative reverse transcription‐PCR and immunofluorescence analysis. Unexpectedly, Schwann cell differentiation appears to proceed normally at the molecular level when monitored by the expression of mRNAs encoding major structural proteins of myelin. Furthermore, an aberrant coexpression of early and late Schwann cell markers was observed. PMP22 itself acquires complex glycosylation, suggesting that trafficking of the myelin protein through the endoplasmic reticulum is not significantly impaired. We suggest that PMP22, when overexpressed, accumulates in a late Golgi‐cell membrane compartment and uncouples myelin assembly from the underlying program of Schwann cell differentiation.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.