Search PubMed⌕ Search

Biomedical subjects

Marcel A Behr

Publications and source records attributed to Marcel A Behr.

At least 19 recordsLinked to original sources
PubMed2006

Mutations in Mycobacterium tuberculosis Rv0444c, the gene encoding anti-SigK, explain high level expression of MPB70 and MPB83 in Mycobacterium bovis.

It has recently been advanced that Mycobacterium tuberculosis sigma factor K (SigK) positively regulates expression of the antigenic proteins MPB70 and MPB83. As expression of these proteins differs between M. tuberculosis (low) and Mycobacterium bovis (high), this study set out to determine whether M. bovis lacks a functional SigK repressor (anti-SigK). By comparing genes near sigK in M. tuberculosis H37Rv and M. bovis AF2122/97, we observed that Rv0444c, annotated as unknown function, had variable sequence in M. bovis. Analysis of in vitro mpt70/mpt83 expression and Rv0444c sequencing across M. tuberculosis complex (MTC) members revealed that high-level expression was associated with a mutated Rv0444c. Complementation of M. bovis bacillus Calmette-Guerin Russia, a high producer of MPB70/MPB83, with wild-type Rv0444c resulted in a significant decrease in mpb70/mpb83 expression. Conversely, a M. tuberculosis H37Rv mutant which expressed sigK but not Rv0444c manifested the M. bovis phenotype of high-level MPB70/MPB83 expression. Further support that Rv0444c encodes the anti-SigK was obtained by yeast two-hybrid studies, where the N-terminal region of Rv0444c-encoded protein interacted with SigK. Together these findings indicate that Rv0444c encodes the regulator of SigK (RskA) and mutations in this gene explain high-level MPT70/MPT83 expression by certain MTC members.

Antigens, Bacterial↗
PubMed2006

Evidence for local transmission and reactivation of tuberculosis in the Toronto Somali community.

To further understand the molecular and clinical epidemiology of tuberculosis in the Toronto Somali community, molecular fingerprinting using IS6110 restriction fragment length polymorphism typing or spoligotyping was performed on M. tuberculosis isolates obtained from Somali-Canadians who developed active disease from 1997 to 2001. Molecular fingerprints were further compared with those obtained from Somalis residing in Denmark. 142 Somali TB patients were reported, for whom, 80 isolates were fingerprinted. 25% of isolates were clustered. Three clusters involving 2 patients each were identified out of the17 isolates that underwent spoligotyping. Of the 63 isolates typed by the IS6110 method, 6 clusters (4 of 2 patients and 2 of 3 patients) were identified. 57% of these isolates were found to be identical to Danish isolates. Our study suggests that a combination of reactivation and recent transmission are responsible for the high incidence rates of tuberculosis in this community. We recommend that ongoing surveillance and treatment programmes be directed towards this community.

Adult↗
PubMed2006

Contact investigation and genotyping to identify tuberculosis transmission to children.

BACKGROUND: Tuberculosis (TB) in young children is an indicator of ongoing community transmission. We examined contact investigations related to pediatric TB, yield for source case identifications and genotypes for relevant Mycobacterium tuberculosis isolates in a low-incidence setting. METHODS: We reviewed public health data for all patients with TB aged <18 years reported to Montreal authorities during 1996 to 2000. M. tuberculosis isolates from patients of all ages were subjected to IS6110-based genotyping, supplemented by spoligotyping, to compare isolates from children and adults during the same years. RESULTS: Sixty-six patients aged <18 years were diagnosed with active TB from 1996 to 2000. Mean age was 11.1 years (standard deviation 6.7 years). Twenty-five children (38%) were Canadian-born, all with at least one foreign-born parent. Nineteen children were diagnosed after contact investigations of known adult cases; 8 underwent no contact investigation. For the remaining 39 children, a total of 616 contacts were identified. The median number of contacts per child was 9 (interquartile range, 6-10). Four hundred eighty-one contacts (78%) underwent tuberculin testing; 188 (39%) were reactors and 186 (39%) began treatment of latent TB. Investigations uncovered 4 probable source cases, all involving parents or other relatives. M. tuberculosis genotyping for 38 children identified up to 14 additional possible source cases; in only one was a possible epidemiologic link evident from public health records. CONCLUSIONS: Among largely foreign-born children with active TB, contact investigations were extensive and often identified latent tuberculosis infection--but rarely source cases. However, genotyping suggested substantial, previously unrecognized transmission to children despite low overall incidence.

Adolescent↗
PubMed2006

Mycobacteria in Crohn's disease: a persistent hypothesis.

Efforts to explore a mycobacterial origin for Crohn's disease typically have involved an epidemiological approach, searching for Mycobacterium avium subsp. paratuberculosis in patient tissue. An alternative approach involves consideration of genetic and experimental data regarding host resistance to mycobacteria. From human and mycobacterial genetics, it is known that mycobacterial diseases depend on both pathogen and host factors and that tuberculosis and leprosy are effectively genetic diseases. The discovery of a number of Crohn's susceptibility genes, including NOD2/CARD15, demonstrates that Crohn's also is a complex genetic disease. Mutations in NOD2/CARD15 do not necessarily lead to Crohn's disease, so other mitigating factors, genetic and/or environmental, probably are required to produce illness. Recent work has shown that NOD2/CARD15 serves a role in bacterial sensing and activation of innate immune responses, providing a link between Crohn's genetics and an environmental factor, potentially a bacterial trigger. In this review, we discuss the current understanding of mycobacterial and Crohn's genetic susceptibility and review the evidence that NOD2/CARD15 may mediate host resistance to mycobacterial infection.

Animals↗
PubMed2006

The two-component regulatory system mtrAB is required for morphotypic multidrug resistance in Mycobacterium avium.

Clinical isolates of the opportunistic pathogen Mycobacterium avium complex (MAC) undergo a reversible switch between red and white colony morphotypes on agar plates containing the lipoprotein stain Congo red. Compared to their isogenic red counterparts, white morphotypic variants are more virulent and more resistant to multiple antibiotics. This report shows that the two-component regulatory system mtrAB is required for the red-to-white switch as well as for other morphotypic switches of MAC. A mutant with a transposon insertion in the histidine protein kinase gene mtrB was isolated from a morphotypically white parent clone. The mutant resembled a naturally occurring red morphotypic variant in that it stained with Congo red, was sensitive to multiple antibiotics, and was permeable by a fluorescent DNA stain. However, it differed from a red variant in that it could not switch to the white or transparent morphotype, and it could not survive intracellularly within macrophage-like cells. Transcomplementation with a cloned wild-type mtrB gene restored to the mutant the ability to form impermeable, drug-resistant white and transparent variants. Quantitative reverse transcriptase PCR showed that mtrB was required for the normal expression of cell surface Mce proteins, some of which are up-regulated in the red-to-white switch. The results indicate that mtrAB functions in regulating the composition and permeability of mycobacterial cell walls and plays a role in the reversible colony type switches of MAC.

ATP-Binding Cassette Transporters↗
PubMed2006

Genetic analysis of Mycobacterium avium complex strains used for producing purified protein derivatives.

For over a century, purified protein derivatives (PPD) have been used to detect mycobacterial infections in humans and livestock. Among these, reagents to detect infections by Mycobacterium avium complex organisms have been produced, but the utility of these reagents has not been clearly established due in part to limited biologic and immunologic standardization. Because there is little information about the strains used to produce these reagents (avian PPD, intracellulare PPD, scrofulaceum PPD, and Johnin), we have performed genetic characterizations of strains used to produce these products. Sequence analysis of 16S rRNA and the hsp65 gene provided results concordant with species designations provided for M. avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum organisms. For M. avium strains, comparative genomic hybridization was performed on a whole-genome DNA microarray, revealing one novel 7.9-kilobase genomic deletion in certain Johnin-producing strains, in addition to genomic variability inherent to the particular M. avium subspecies. Our findings indicate that considerable genomic differences exist between organisms used for reagents and the infecting organism being studied. These results serve as a baseline for potency studies of different preparations and should aid in comparative studies of newly discovered antigens for the diagnosis of infection and disease by M. avium complex organisms.

Animals↗
PubMed2006

Evaluation of in situ methods used to detect Mycobacterium avium subsp. paratuberculosis in samples from patients with Crohn's disease.

In common with other diagnostic tests, detection of mycobacteria in tissue by microscopic examination is susceptible to spectrum bias. Since Crohn's disease is defined by the absence of detectable pathogenic organisms, the use of in situ techniques to search for Mycobacterium avium subsp. paratuberculosis in Crohn's disease samples requires validation of methods in a paucibacillary setting. To generate paucibacillary infection, C57BL/6 mice were artificially infected with Mycobacterium avium subsp. paratuberculosis strain K10 and M. tuberculosis H37Rv, yielding tissues harboring fewer than one bacillus per oil immersion field. Serial sections of organs were then studied by cell wall-based staining techniques (Ziehl-Neelsen and auramine rhodamine) and nucleic acid-based staining techniques (in situ hybridization [ISH] and indirect in situ PCR [IS PCR]). Microscopic examination and measurement of morphometric parameters of bacilli revealed that for all methods, Mycobacterium avium subsp. paratuberculosis bacilli were observed to be shorter, smaller, and less rod shaped than M. tuberculosis bacilli. Ziehl-Neelsen, auramine rhodamine stains, ISH targeting rRNA, and IS-PCR targeting the IS900 element afforded comparable sensitivities, but for all methods, visualization of individual bacterial forms required magnification x1,000. Auramine rhodamine staining and IS-PCR generated positive signals in negative controls, indicating the nonspecificity of these assays. Together, our results indicate that detection of Mycobacterium avium subsp. paratuberculosis bacilli in tissue requires oil immersion microscopy, that rRNA-ISH provides sensitivity and specificity comparable to those of Ziehl-Neelsen staining, and that the microscopic detection limit for Mycobacterium avium subsp. paratuberculosis in tissue is governed more by bacterial burden than by staining method.

Animals↗
PubMed2006

Sequencing of hsp65 distinguishes among subsets of the Mycobacterium avium complex.

The Mycobacterium avium complex consists of epidemiologically distinct subsets. The classification of these subsets is complicated by a number of factors, including the ambiguous results obtained with phenotypic and genetic assays and the recent appreciation that human and avian strains appear to be distinct. In previous work, sequencing based on a 441-bp portion of the hsp65 gene has proven to efficiently classify isolates within the Mycobacterium genus but provides low resolution for distinguishing among members of the M. avium complex. Therefore, in this study, we have targeted the more variable 3' region of the hsp65 gene to determine whether it can effectively discriminate M. avium complex isolates at the levels of species and subspecies. Primers designed for this target consistently generated amplicons for all organisms classified as M. avium complex. Sequences obtained indicate that M. intracellulare is genetically divergent from M. avium organisms, and distinct sequevars were obtained for M. avium subsets, including M. avium subsp. avium (bird type), M. avium subsp. hominissuis, and M. avium subsp. paratuberculosis. In addition, sequence differences served to distinguish bovine from ovine strains of M. avium subsp. paratuberculosis. A unique profile for M. avium subsp. silvaticum was not obtained. These results indicate that sequencing the 3' region of the hsp65 gene can simply and unambiguously distinguish species and subspecies of the M. avium complex.

Animals↗
PubMed2006

Insertion sequence IS900 revisited.

Many studies investigating Mycobacterium avium subsp. paratuberculosis in Crohn's disease have used molecular detection of IS900 in clinical samples, but some have described polymorphisms in IS900 as variants of this organism. Analysis of 23 M. avium subsp. paratuberculosis isolates revealed that IS900 is highly conserved, with only two sequevars distinguishing sheep and cattle lineages. Amplification of IS900-like sequences is not sufficient as a proxy for M. avium subsp. paratuberculosis.

Animals↗
PubMed2006

Isolation of the genome sequence strain Mycobacterium avium 104 from multiple patients over a 17-year period.

The genome sequence strain 104 of the opportunistic pathogen Mycobacterium avium was isolated from an adult AIDS patient in Southern California in 1983. Isolates of non-paratuberculosis M. avium from 207 other patients in Southern California and elsewhere were examined for genotypic identity to strain 104. This process was facilitated by the use of a novel two-step approach. In the first step, all 208 strains in the sample were subjected to a high-throughput, large sequence polymorphism (LSP)-based genotyping test, in which DNA from each strain was tested by PCR for the presence or absence of 4 hypervariable genomic regions. Nineteen isolates exhibited an LSP type that resembled that of strain 104. This subset of 19 isolates was then subjected to high-resolution repetitive sequence-based PCR typing, which identified 10 isolates within the subset that were genotypically identical to strain 104. These isolates came from 10 different patients at 5 clinical sites in the western United States, and they were isolated over a 17-year time span. Therefore, the sequenced genome of M. avium strain 104 has been associated with disease in multiple patients in the western United States. Although M. avium is known for its genetic plasticity, these observations also show that strains of the pathogen can be genotypically stable over extended time periods.

Bacterial Typing Techniques↗
PubMed2006

Differentiating host-associated variants of Mycobacterium avium by PCR for detection of large sequence polymorphisms.

The Mycobacterium avium species consists of a group of organisms that are genetically related but phenotypically diverse, with certain variants presenting clear differences in terms of their host association and disease manifestations. The ability to distinguish between these subtypes is of relevance for accurate diagnosis and for control programs. Using a comparative genomics approach, we have uncovered large sequence polymorphisms that are, respectively, absent from bird-type M. avium isolates and from cattle types and sheep types of M. avium subsp. paratuberculosis. By evaluating the distribution of these genomic polymorphisms across a panel of strains, we were able to assign unique genomic signatures to these host-associated variants. We propose a simple PCR-based strategy based on these polymorphisms that can rapidly type M. avium isolates into these subgroups.

Animals↗
PubMed2005

Rate of reinfection tuberculosis after successful treatment is higher than rate of new tuberculosis.

RATIONALE: In a high-tuberculosis (TB) incidence area of Cape Town, South Africa, there is a very high rate of unexplained recurrent TB. The incidence of new bacteriologically confirmed disease in the area is 313 per 100,000 individuals. OBJECTIVE: To estimate the rate of recurrent TB attributable to reinfection after successful treatment. METHODS: All patients with reported TB in the area between 1993 and 1998 were followed up to 2001 for disease needing retreatment (recurrences). Patients who were multi-drug-resistant or who had treatment failure, were transferred, or died during treatment were excluded. Analysis was restricted to patients for whom DNA fingerprinting of their Mycobacterium tuberculosis isolates was obtained. Reinfection TB was defined as a recurrent TB episode in which the strains of the separate episodes differed by more than four bands. MEASUREMENTS AND MAIN RESULTS: 612 of 897 (68%) patients had a DNA fingerprint available at enrollment. Median duration of follow-up was 5.2 years. Recurrent TB occurred in 108 of 612 (18%) patients, of whom 61 of 447 (14%) experienced recurrence after successful treatment, and 47 of 165 (28%) experience recurrence after default. Of the 108 patients with recurrent TB, 68 (63%) had a DNA fingerprint in the second episode. Among these patients, 24 of 31 (77%) recurrences after successful treatment and 4 of 37 (11%) recurrences after default were attributable to reinfection. The reinfection disease rate after successful treatment was estimated at 2.2 per 100 person-years. CONCLUSIONS: The age-adjusted incidence rate of TB attributable to reinfection after successful treatment was four times that of new TB. People who had TB once are at a strongly increased risk of developing TB when reinfected.

Adolescent↗
PubMed2005

The origin and evolution of Mycobacterium tuberculosis.

This article introduces the tools and terminology used for the classification of specific isolates of the Mycobacterium tuberculosis complex (MTC). The utility of these tools and terminology is illustrated by discussing work from independent laboratories that have established a genome-based phylogeny for the MTC. It considers the use of these markers to distinguish atypical isolates not conforming to attributes of traditional MTC members. Finally, it discusses the current genomic evidence regarding the origin and evolution of M. tuberculosis in the context of its relevance for tuberculosis control in humans and other mammalian hosts.

Animals↗
PubMed2005

Point mutations in the DNA- and cNMP-binding domains of the homologue of the cAMP receptor protein (CRP) in Mycobacterium bovis BCG: implications for the inactivation of a global regulator and strain attenuation.

The genome of Mycobacterium tuberculosis H37Rv includes a homologue of the CRP/FNR (cAMP receptor protein/fumarate and nitrate reduction regulator) family of transcription regulators encoded by Rv3676. Sequencing of the orthologous gene from attenuated Mycobacterium bovis Bacille Calmette-Guérin (BCG) strains revealed point mutations that affect the putative DNA-binding and cNMP-binding domains of the encoded protein. These mutations are not present in the published sequences of the Rv3676 orthologues in M. bovis, M. tuberculosis or Mycobacterium leprae. An Escherichia coli lacZ reporter system was used to show that the M. tuberculosis Rv3676 protein binds to DNA sites for CRP, but this DNA binding was decreased or abolished with the Rv3676 protein counterparts from BCG strains. The DNA-binding ability of the M. tuberculosis Rv3676 protein was decreased by the introduction of base changes corresponding to the BCG point mutations. Conversely, the DNA binding of the BCG Rv3676 proteins from BCG strains was restored by removing the mutations. These data show that in this reporter system the point mutations present in the Rv3676 orthologue in BCG strains render its function defective (early strains) or abolished (late strains) and suggest that this protein might be naturally defective in M. bovis BCG strains. This raises the possibility that a contributing factor to the attenuation of BCG strains may be an inability of this global regulator to control the expression of genes required for in vivo survival and persistence.

Amino Acid Sequence↗
PubMed2005

Reduced expression of antigenic proteins MPB70 and MPB83 in Mycobacterium bovis BCG strains due to a start codon mutation in sigK.

Mycobacterium bovis Bacille Calmette-Guerin (BCG) strains are genetically and phenotypically heterogeneous. Expression of the antigenic proteins MPB70 and MPB83 is known to vary considerably across BCG strains; however, the reason for this phenotypic difference has remained unknown. By immunoblot, we separated BCG into high- and low-producing strains. By quantitative reverse transcription polymerase chain reaction (RT-PCR), we determined that transcription of the antigen-encoding genes, mpb70 and mpb83, follows the same strain pattern with mRNA levels reduced over 50-fold in low-producing strains. Transcriptome comparison of the same BCG strains by DNA microarray revealed two gene regions consistently downregulated in low-producing strains compared with high-producing strains, one including mpb70 (Rv2875) and mpb83 (Rv2873) and a second that includes the predicted sigma factor, sigK. DNA sequence analysis revealed a point mutation in the start codon of sigK in all low-producing BCG strains. Complementation of a low-producing strain, BCG Pasteur, with wild-type sigK fully restored MPB70 and MPB83 production. Microarray-based analysis and confirmatory RT-PCR of the complemented strains revealed an upregulation in gene transcription limited to the sigK and the mpb83/mpb70 gene regions. These data demonstrate that a mutation of sigK is responsible for decreased expression of MPB70 and MPB83 in low-producing BCG strains and provide clues into the role of Mycobacterium tuberculosis SigK.

Antigens, Bacterial↗
PubMed2005

Revisiting the evolution of Mycobacterium bovis.

Though careful consideration has been placed towards genetic characterization of tubercle bacillus isolates causing disease in humans, those causing disease predominantly among wild and domesticated mammals have received less attention. In contrast to Mycobacterium tuberculosis, whose host range is largely specific to humans, M. bovis and "M bovis-like" organisms infect a broad range of animal species beyond their most prominent host in cattle. To determine whether strains of variable genomic content are associated with distinct distributions of disease, the DNA contents of M. bovis or M. bovis-like isolates from a variety of hosts were investigated via Affymetrix GeneChip. Consistent with previous genomic analysis of the M. tuberculosis complex (MTC), large sequence polymorphisms of putative diagnostic and biological consequence were able to unambiguously distinguish interrogated isolates. The distribution of deleted regions indicates organisms genomically removed from M. bovis and also points to structured genomic variability within M. bovis. Certain genomic profiles spanned a variety of hosts but were clustered by geography, while others associated primarily with host type. In contrast to the prevailing assumption that M. bovis has broad host capacity, genomic profiles suggest that distinct MTC lineages differentially infect a variety of mammals. From this, a phylogenetic stratification of genotypes offers a predictive framework upon which to base future genetic and phenotypic studies of the MTC.

Evolution, Molecular↗
PubMed2005

Sensitivities and specificities of spoligotyping and mycobacterial interspersed repetitive unit-variable-number tandem repeat typing methods for studying molecular epidemiology of tuberculosis.

The development of PCR-based genotyping modalities (spoligotyping and mycobacterial interspersed repetitive unit-variable-number tandem repeat [MIRU-VNTR] typing) offers promise for real-time molecular epidemiological studies of tuberculosis (TB). However, the utility of these methods depends on their capacity to appropriately classify isolates. To determine the operating parameters of spoligotyping and MIRU-VNTR typing, we have compared results generated by these newer tests to the standard typing method, IS6110 restriction fragment length polymorphism, in analyses restricted to high-copy-number IS6110 isolates. Sensitivities of the newer tests were estimated as the percentages of isolates with identical IS6110 fingerprints that had identical spoligotypes and MIRU-VNTR types. The specificities of these tests were estimated as the percentages of isolates with unique IS6110 fingerprints that had unique spoligotypes and MIRU-VNTR types. The sensitivity of MIRU-VNTR typing was 52% (95% confidence interval [CI], 31 to 72%), and the sensitivity of spoligotyping was 83% (95% CI, 63 to 95%). The specificity of MIRU-VNTR typing was 56% (95% CI, 51 to 62%), and the specificity of spoligotyping was 40% (95% CI, 35 to 46%). The proportion of isolates estimated to be due to recent transmission was 4% by identical IS6110 patterns, 19% by near-identical IS6110 patterns, 33% by MIRU-VNTR typing, and 53% by spoligotyping. The low calculated specificities of spoligotyping and MIRU-VNTR typing led to misclassification of cases, inflated estimates of TB transmission, and low positive predictive values, suggesting that these techniques have unsuitable operating parameters for population-based molecular epidemiology studies.

Bacterial Typing Techniques↗