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1.  Does Choice Matter? Reference-Based Alignment for Molecular Epidemiology of Tuberculosis 
Journal of Clinical Microbiology  2016;54(7):1891-1895.
When using genome sequencing for molecular epidemiology, short sequence reads are aligned to an arbitrary reference strain to detect single nucleotide polymorphisms. We investigated whether reference genome selection influences epidemiological inferences of Mycobacterium tuberculosis transmission by aligning sequence reads from 162 closely related lineage 4 (Euro-American) isolates to 7 different genomes. Phylogenetic trees were consistent with use of all but the most divergent genomes, suggesting that reference choice can be based on considerations other than M. tuberculosis lineage.
doi:10.1128/JCM.00364-16
PMCID: PMC4922084  PMID: 27076659
2.  Alu repeats as transcriptional regulatory platforms in macrophage responses to M. tuberculosis infection 
Nucleic Acids Research  2016;44(22):10571-10587.
To understand the epigenetic regulation of transcriptional response of macrophages during early-stage M. tuberculosis (Mtb) infection, we performed ChIPseq analysis of H3K4 monomethylation (H3K4me1), a marker of poised or active enhancers. De novo H3K4me1 peaks in infected cells were associated with genes implicated in host defenses and apoptosis. Our analysis revealed that 40% of de novo regions contained human/primate-specific Alu transposable elements, enriched in the AluJ and S subtypes. These contained several transcription factor binding sites, including those for members of the MEF2 and ATF families, and LXR and RAR nuclear receptors, all of which have been implicated in macrophage differentiation, survival, and responses to stress and infection. Combining bioinformatics, molecular genetics, and biochemical approaches, we linked genes adjacent to H3K4me1-associated Alu repeats to macrophage metabolic responses against Mtb infection. In particular, we show that LXRα signaling, which reduced Mtb viability 18-fold by altering cholesterol metabolism and enhancing macrophage apoptosis, can be initiated at response elements present in Alu repeats. These studies decipher the mechanism of early macrophage transcriptional responses to Mtb, highlighting the role of Alu element transposition in shaping human transcription programs during innate immunity.
doi:10.1093/nar/gkw782
PMCID: PMC5159539  PMID: 27604870
3.  Housing and tuberculosis in an Inuit village in northern Quebec: a case-control study 
CMAJ Open  2016;4(3):E496-E506.
Background:
Between November 2011 and November 2012, an Inuit village in Nunavik, Quebec experienced a surge in the occurrence of active TB; contact investigations showed that TB infection was highly prevalent (62.6%), particularly among those over age 14 years (78.8%). A nested case-control study showed that nutritional inadequacy was associated with acquisition of infection but not progression to disease. We performed a study to determine whether characteristics of one's dwelling were associated with 1) acquisition of newly diagnosed TB infection and 2) progression to confirmed or probable disease among those with TB infection.
Methods:
In this nested case-control study, we enrolled 200 people who were household or social contacts of at least 1 person with active TB or had received a diagnosis of active TB and assessed whether characteristics of their dwellings were associated with their odds of having newly diagnosed TB infection and/or odds of progression to disease between November 2011 and November 2012. For our first objective, we compared participants with newly diagnosed TB infection (regardless of their disease status) to a control group of contacts who were uninfected. For the second objective, we compared participants with confirmed or probable disease to a control group consisting of those with infection but no disease. We used information collected during investigation of the contacts and from study questionnaires to determine whether participants may have been exposed to TB in their own home (if they had shared a dwelling with someone who had smear-positive TB during the outbreak) or in other dwellings that they visited at least weekly.
Results:
The participants lived in 79 dwellings. The mean number of people per room was 1.1 (standard deviation [SD] 0.5). The mean room size and ventilation level of the common living space (kitchen and living/dining rooms) were 67.9 (SD 9.4) m3 and 1.69 (SD 0.26) air changes per hour, respectively. After adjustment for potential confounders, the number of people per room was positively associated with the odds of newly diagnosed infection and odds of disease, but only among participants who lived with someone with smear-positive TB (the minority of participants). Other dwelling characteristics were not associated with either outcome.
Interpretation:
Reducing household crowding may contribute to TB prevention. Overall, our investigations have not identified associations that explain the elevated disease risk in this village. In light of our results and considering the high prevalence of TB infection, treatment of latent infection is an essential intervention for long-term reduction of TB incidence in this village.
doi:10.9778/cmajo.20160049
PMCID: PMC5047805  PMID: 27730114
4.  The implications of whole-genome sequencing in the control of tuberculosis 
The availability of whole-genome sequencing (WGS) as a tool for the diagnosis and clinical management of tuberculosis (TB) offers considerable promise in the fight against this stubborn epidemic. However, like other new technologies, the best application of WGS remains to be determined, for both conceptual and technical reasons. In this review, we consider the potential value of WGS in the clinical laboratory for the detection of Mycobacterium tuberculosis and the prediction of antibiotic resistance. We also discuss issues pertaining to data generation, interpretation and dissemination, given that WGS has to date been generally performed in research labs where results are not necessarily packaged in a clinician-friendly format. Although WGS is far more accessible now than it was in the past, the transition from a research tool to study TB into a clinical test to manage this disease may require further fine-tuning. Improvements will likely come through iterative efforts that involve both the laboratories ready to move TB into the genomic era and the front-line clinical/public health staff who will be interpreting the results to inform management decisions.
doi:10.1177/2049936115624630
PMCID: PMC4784569  PMID: 27034776
clinical microbiology; diagnostics; drug resistance; Mycobacterium tuberculosis; whole-genome sequencing
5.  Iron Acquisition in Mycobacterium avium subsp. paratuberculosis 
Journal of Bacteriology  2016;198(5):857-866.
ABSTRACT
Mycobacterium avium subsp. paratuberculosis is a host-adapted pathogen that evolved from the environmental bacterium M. avium subsp. hominissuis through gene loss and gene acquisition. Growth of M. avium subsp. paratuberculosis in the laboratory is enhanced by supplementation of the media with the iron-binding siderophore mycobactin J. Here we examined the production of mycobactins by related organisms and searched for an alternative iron uptake system in M. avium subsp. paratuberculosis. Through thin-layer chromatography and radiolabeled iron-uptake studies, we showed that M. avium subsp. paratuberculosis is impaired for both mycobactin synthesis and iron acquisition. Consistent with these observations, we identified several mutations, including deletions, in M. avium subsp. paratuberculosis genes coding for mycobactin synthesis. Using a transposon-mediated mutagenesis screen conditional on growth without myobactin, we identified a potential mycobactin-independent iron uptake system on a M. avium subsp. paratuberculosis-specific genomic island, LSPP15. We obtained a transposon (Tn) mutant with a disruption in the LSPP15 gene MAP3776c for targeted study. The mutant manifests increased iron uptake as well as intracellular iron content, with genes downstream of the transposon insertion (MAP3775c to MAP3772c [MAP3775-2c]) upregulated as the result of a polar effect. As an independent confirmation, we observed the same iron uptake phenotypes by overexpressing MAP3775-2c in wild-type M. avium subsp. paratuberculosis. These data indicate that the horizontally acquired LSPP15 genes contribute to iron acquisition by M. avium subsp. paratuberculosis, potentially allowing the subsequent loss of siderophore production by this pathogen.
IMPORTANCE Many microbes are able to scavenge iron from their surroundings by producing iron-chelating siderophores. One exception is Mycobacterium avium subsp. paratuberculosis, a fastidious, slow-growing animal pathogen whose growth needs to be supported by exogenous mycobacterial siderophore (mycobactin) in the laboratory. Data presented here demonstrate that, compared to other closely related M. avium subspecies, mycobactin production and iron uptake are different in M. avium subsp. paratuberculosis, and these phenotypes may be caused by numerous deletions in its mycobactin biosynthesis pathway. Using a genomic approach, supplemented by targeted genetic and biochemical studies, we identified that LSPP15, a horizontally acquired genomic island, may encode an alternative iron uptake system. These findings shed light on the potential physiological consequence of horizontal gene transfer in M. avium subsp. paratuberculosis evolution.
doi:10.1128/JB.00922-15
PMCID: PMC4810606  PMID: 26712939
6.  Genomic expression catalogue of a global collection of BCG vaccine strains show evidence for highly diverged metabolic and cell-wall adaptations 
Scientific Reports  2015;5:15443.
Although Bacillus Calmette-Guérin (BCG) vaccines against tuberculosis have been available for more than 90 years, their effectiveness has been hindered by variable protective efficacy and a lack of lasting memory responses. One factor contributing to this variability may be the diversity of the BCG strains that are used around the world, in part from genomic changes accumulated during vaccine production and their resulting differences in gene expression. We have compared the genomes and transcriptomes of a global collection of fourteen of the most widely used BCG strains at single base-pair resolution. We have also used quantitative proteomics to identify key differences in expression of proteins across five representative BCG strains of the four tandem duplication (DU) groups. We provide a comprehensive map of single nucleotide polymorphisms (SNPs), copy number variation and insertions and deletions (indels) across fourteen BCG strains. Genome-wide SNP characterization allowed the construction of a new and robust phylogenic genealogy of BCG strains. Transcriptional and proteomic profiling revealed a metabolic remodeling in BCG strains that may be reflected by altered immunogenicity and possibly vaccine efficacy. Together, these integrated-omic data represent the most comprehensive catalogue of genetic variation across a global collection of BCG strains.
doi:10.1038/srep15443
PMCID: PMC4614345  PMID: 26487098
7.  Pre-Columbian mycobacterial genomes reveal seals as a source of New World human tuberculosis 
Nature  2014;514(7523):494-497.
Modern strains of Mycobacterium tuberculosis from the Americas are closely related to those from Europe, supporting the assumption that human tuberculosis was introduced post-contact1. This notion, however, is incompatible with archaeological evidence of pre-contact tuberculosis in the New World2. Comparative genomics of modern isolates suggests that M. tuberculosis attained its worldwide distribution following human dispersals out of Africa during the Pleistocene epoch3, although this has yet to be confirmed with ancient calibration points. Here we present three 1,000-year-old mycobacterial genomes from Peruvian human skeletons, revealing that a member of the M. tuberculosis complex caused human disease before contact. The ancient strains are distinct from known human-adapted forms and are most closely related to those adapted to seals and sea lions. Two independent dating approaches suggest a most recent common ancestor for the M. tuberculosis complex less than 6,000 years ago, which supports a Holocene dispersal of the disease. Our results implicate sea mammals as having played a role in transmitting the disease to humans across the ocean.
doi:10.1038/nature13591
PMCID: PMC4550673  PMID: 25141181
8.  CARD9 Deficiency and Spontaneous Central Nervous System Candidiasis: Complete Clinical Remission With GM-CSF Therapy 
We demonstrate autosomal-recessive CARD9 deficiency in a patient with relapsing Candida albicans meningoencephalitis. The novel, hypomorphic mutation impaired granulocyte-macrophage colony-stimulating factor (GM-CSF) but not Th17 responses. Adjunctive GM-CSF therapy resulted in clinical remission, suggesting that a CARD9/GM-CSF axis contributes to susceptibility to candidiasis.
We demonstrate autosomal-recessive Caspase Recruitment Domain-containing protein 9 (CARD9) deficiency in a patient with relapsing C. albicans meningoencephalitis. We identified a novel, hypomorphic mutation with intact Th17 responses, but impaired GM-CSF responses. We report complete clinical remission with adjunctive GM-CSF therapy, suggesting that a CARD9/GM-CSF axis contributes to susceptibility to candidiasis.
doi:10.1093/cid/ciu215
PMCID: PMC4305130  PMID: 24704721
CARD9; spontaneous central nervous system candidiasis; Candida; GM-CSF; Th17
9.  Origins of a 350-Kilobase Genomic Duplication in Mycobacterium tuberculosis and Its Impact on Virulence 
Infection and Immunity  2014;82(7):2902-2912.
In the present study, we have investigated the evolution and impact on virulence of a 350-kb genomic duplication present in the most recently evolved members of the Mycobacterium tuberculosis East Asian lineage. In a mouse model of infection, comparing HN878 subclones HN878-27 (no duplication) and HN878-45 (with the 350-kb duplication) revealed that the latter is impaired for in vivo growth during the initial 3 weeks of infection. Furthermore, the median survival time of mice infected with isolate HN878-45 is significantly longer (77 days) than that of mice infected with HN878-27. Whole-genome sequencing of both isolates failed to reveal any mutational events other than the duplication that could account for such a substantial difference in virulence. Although we and others had previously speculated that the 350-kb duplication arose in response to some form of host-applied selective pressure (P. Domenech, G. S. Kolly, L. Leon-Solis, A. Fallow, M. B. Reed, J. Bacteriol. 192:4562–4570, 2010, and B. Weiner, J. Gomez, T. C. Victor, R. M. Warren, A. Sloutsky, B. B. Plikaytis, J. E. Posey, P. D. van Helden, N. C. Gey van Pittius, M. Koehrsen, P. Sisk, C. Stolte, J. White, S. Gagneux, B. Birren, D. Hung, M. Murray, J. Galagan, PLoS One 7:e26038, 2012), here we show that this large chromosomal amplification event is very rapidly selected within standard in vitro broth cultures in a range of isolates. Indeed, subclones harboring the duplication were detectable after just five rounds of in vitro passage. In contrast, the duplication appears to be highly unstable in vivo and is negatively selected during the later stages of infection in mice. We believe that the rapid in vitro evolution of M. tuberculosis is an underappreciated aspect of its biology that is often ignored, despite the fact that it has the potential to confound the data and conclusions arising from comparative studies of isolates at both the genotypic and phenotypic levels.
doi:10.1128/IAI.01791-14
PMCID: PMC4097636  PMID: 24778110
10.  Contribution of increased ISG15, ISGylation and deregulated Type I IFN signaling in Usp18 mutant mice during the course of bacterial infections 
Genes and immunity  2014;15(5):282-292.
Host genetics plays a key role in susceptibility to Salmonella Typhimurium infection. We previously used N-ethyl-N-nitrosourea (ENU) mutagenesis to identify a loss of function mutation within the gene ubiquitin specific peptidase 18 (Usp18Ity9), which confers increased susceptibility to Salmonella Typhimurium. USP18 functions to regulate type I IFN signaling and as a protease to remove ISG15 from substrate proteins. Usp18Ity9 mice are susceptible to infection with Salmonella Typhimurium and have increased expression and function of ISG15, but Usp18Ity9 mice lacking Isg15 do not show improved survival with Salmonella challenge. Type I IFN signaling is increased in Usp18Ity9 mice and inhibition of type I IFN signaling is associated with improved survival in mutant mice. Hyperactivation of type I IFN signaling leads to increased IL-10, deregulated expression of autophagy markers, and elevated IL-1β and IL-17. Furthermore, Usp18Ity9 mice are more susceptible to infection with Mycobacterium tuberculosis, have increased bacterial load in lung and spleen, elevated inflammatory cytokines and more severe lung pathology. These findings demonstrate that regulation of type I IFN signaling is the predominant mechanism affecting the susceptibility of Usp18Ity9 mice to Salmonella infection and that hyperactivation of signaling leads to increased IL-10, deregulation of autophagic markers and increased proinflammatory cytokine production.
doi:10.1038/gene.2014.17
PMCID: PMC4111656  PMID: 24807690 CAMSID: cams4329
USP18; innate immunity; Salmonella; mycobacteria; type I IFN; ISGylation; autophagy
11.  Quantiferon Gold-in-tube assay for TB screening in HIV infected children: influence of quantitative values 
BMC Infectious Diseases  2014;14:516.
Background
HIV infected children are at increased risk of TB disease and require annual TB screening. Data on use of IGRA for TB screening in them are limited. We retrospectively evaluated the usefulness of Quantiferon Gold-in-tube test (QFT), an IGRA in screening for LTBI in relatively healthy, immunologically stable HIV infected children.
Methods
HIV infected children with no prior history of TB were screened for latent TB as part of routine care. They underwent risk of TB assessment, TST and QFT. QFT was repeated twice or three times depending on the quantitative values. Independent test validation was also performed.
Results
Eighty one children had 109 QFT tests. All had adequate mitogen responses. The initial QFT was positive in 15 (18.5%) children; quantitative IGRA responses were 0.35-1.0 IU/mL in 9 (60%), 1.0-10 IU/mL in5 (33.3%) and >10 IU/mL in 1 (6.7%). None that tested positive had documented TB exposure or TB disease. Baseline characteristics in the QFT positive and negative groups were similar. Repeat testing within 17 weeks demonstrated reversion to negative in 79% of cases. Repeat blinded independent testing of all QFT positive results and a random selection of initial negative tests demonstrated concordance in 96% of cases. Seven children (QFT > 1.0 IU/mL or positive TST) were offered INH preventive therapy. In no case has TB disease developed in 2 years of close follow-up.
Conclusions
QFT is a valid method for LTBI screening relatively healthy, immunologically stable HIV infected children. However, reversion to negative on repeat testing and lack of correlation with TST results and risk of TB exposure makes interpretation difficult.
doi:10.1186/1471-2334-14-516
PMCID: PMC4181619  PMID: 25248406
HIV; Tuberculosis; Screening; IGRA; Quantiferon
12.  Disruption of Mycobacterium avium subsp. paratuberculosis-specific genes impairs in vivo fitness 
BMC Genomics  2014;15(1):415.
Background
Mycobacterium avium subsp. paratuberculosis (MAP) is an obligate intracellular pathogen that infects many ruminant species. The acquisition of foreign genes via horizontal gene transfer has been postulated to contribute to its pathogenesis, as these genetic elements are absent from its putative ancestor, M. avium subsp. hominissuis (MAH), an environmental organism with lesser pathogenicity. In this study, high-throughput sequencing of MAP transposon libraries were analyzed to qualitatively and quantitatively determine the contribution of individual genes to bacterial survival during infection.
Results
Out of 52384 TA dinucleotides present in the MAP K-10 genome, 12607 had a MycoMarT7 transposon in the input pool, interrupting 2443 of the 4350 genes in the MAP genome (56%). Of 96 genes situated in MAP-specific genomic islands, 82 were disrupted in the input pool, indicating that MAP-specific genomic regions are dispensable for in vitro growth (odds ratio = 0.21). Following 5 independent in vivo infections with this pool of mutants, the correlation between output pools was high for 4 of 5 (R = 0.49 to 0.61) enabling us to define genes whose disruption reproducibly reduced bacterial fitness in vivo. At three different thresholds for reduced fitness in vivo, MAP-specific genes were over-represented in the list of predicted essential genes. We also identified additional genes that were severely depleted after infection, and several of them have orthologues that are essential genes in M. tuberculosis.
Conclusions
This work indicates that the genetic elements required for the in vivo survival of MAP represent a combination of conserved mycobacterial virulence genes and MAP-specific genes acquired via horizontal gene transfer. In addition, the in vitro and in vivo essential genes identified in this study may be further characterized to offer a better understanding of MAP pathogenesis, and potentially contribute to the discovery of novel therapeutic and vaccine targets.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-415) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-415
PMCID: PMC4058006  PMID: 24885784
Mycobacterium avium; M. avium subsp. paratuberculosis; Transposon insertion sequencing; Horizontal gene transfer; Mycobacterial pathogenesis
13.  Novel Feature of Mycobacterium avium subsp. paratuberculosis, Highlighted by Characterization of the Heparin-Binding Hemagglutinin Adhesin 
Journal of Bacteriology  2013;195(21):4844-4853.
Mycobacterium avium subsp. paratuberculosis comprises two genotypically defined groups, known as the cattle (C) and sheep (S) groups. Recent studies have reported phenotypic differences between M. avium subsp. paratuberculosis groups C and S, including growth rates, infectivity for macrophages, and iron metabolism. In this study, we investigated the genotypes and biological properties of the virulence factor heparin-binding hemagglutinin adhesin (HBHA) for both groups. In Mycobacterium tuberculosis, HBHA is a major adhesin involved in mycobacterium-host interactions and extrapulmonary dissemination of infection. To investigate HBHA in M. avium subsp. paratuberculosis, we studied hbhA polymorphisms by fragment analysis using the GeneMapper technology across a large collection of isolates genotyped by mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) and IS900 restriction fragment length polymorphism (RFLP-IS900) analyses. Furthermore, we analyzed the structure-function relationships of recombinant HBHA proteins of types C and S by heparin-Sepharose chromatography and surface plasmon resonance (SPR) analyses. In silico analysis revealed two forms of HBHA, corresponding to the prototype genomes for the C and S types of M. avium subsp. paratuberculosis. This observation was confirmed using GeneMapper on 85 M. avium subsp. paratuberculosis strains, including 67 strains of type C and 18 strains of type S. We found that HBHAs from all type C strains contain a short C-terminal domain, while those of type S present a long C-terminal domain, similar to that produced by Mycobacterium avium subsp. avium. The purification of recombinant HBHA from M. avium subsp. paratuberculosis of both types by heparin-Sepharose chromatography highlighted a correlation between their affinities for heparin and the lengths of their C-terminal domains, which was confirmed by SPR analysis. Thus, types C and S of M. avium subsp. paratuberculosis may be distinguished by the types of HBHA they produce, which differ in size and adherence properties, thereby providing new evidence that strengthens the genotypic differences between the C and S types of M. avium subsp. paratuberculosis.
doi:10.1128/JB.00671-13
PMCID: PMC3807500  PMID: 23974028
14.  Building a better bacillus: the emergence of Mycobacterium tuberculosis 
The genus Mycobacterium is comprised of more than 150 species that reside in a wide variety of habitats. Most mycobacteria are environmental organisms that are either not associated with disease or are opportunistic pathogens that cause non-transmissible disease in immunocompromised individuals. In contrast, a small number of species, such as the tubercle bacillus, Mycobacterium tuberculosis, are host-adapted pathogens for which there is no known environmental reservoir. In recent years, gene disruption studies using the host-adapted pathogen have uncovered a number of “virulence factors,” yet genomic data indicate that many of these elements are present in non-pathogenic mycobacteria. This suggests that much of the genetic make-up that enables virulence in the host-adapted pathogen is already present in environmental members of the genus. In addition to these generic factors, we hypothesize that molecules elaborated exclusively by professional pathogens may be particularly implicated in the ability of M. tuberculosis to infect, persist, and cause transmissible pathology in its host species, Homo sapiens. One approach to identify these molecules is to employ comparative analysis of mycobacterial genomes, to define evolutionary events such as horizontal gene transfer (HGT) that contributed M. tuberculosis-specific genetic elements. Independent studies have now revealed the presence of HGT genes in the M. tuberculosis genome and their role in the pathogenesis of disease is the subject of ongoing investigations. Here we review these studies, focusing on the hypothesized role played by HGT loci in the emergence of M. tuberculosis from a related environmental species into a highly specialized human-adapted pathogen.
doi:10.3389/fmicb.2014.00139
PMCID: PMC3982062  PMID: 24765091
Mycobacterium tuberculosis; Mycobacterium kansasii; M. tuberculosis-specific genes; horizontal gene transfer; comparative genomics
15.  atpE gene as a new useful specific molecular target to quantify Mycobacterium in environmental samples 
BMC Microbiology  2013;13:277.
Background
The environment is the likely source of many pathogenic mycobacterial species but detection of mycobacteria by bacteriological tools is generally difficult and time-consuming. Consequently, several molecular targets based on the sequences of housekeeping genes, non-functional RNA and structural ribosomal RNAs have been proposed for the detection and identification of mycobacteria in clinical or environmental samples. While certain of these targets were proposed as specific for this genus, most are prone to false positive results in complex environmental samples that include related, but distinct, bacterial genera. Nowadays the increased number of sequenced genomes and the availability of software for genomic comparison provide tools to develop novel, mycobacteria-specific targets, and the associated molecular probes and primers. Consequently, we conducted an in silico search for proteins exclusive to Mycobacterium spp. genomes in order to design sensitive and specific molecular targets.
Results
Among the 3989 predicted proteins from M. tuberculosis H37Rv, only 11 proteins showed 80% to 100% of similarity with Mycobacterium spp. genomes, and less than 50% of similarity with genomes of closely related Corynebacterium, Nocardia and Rhodococcus genera. Based on DNA sequence alignments, we designed primer pairs and a probe that specifically detect the atpE gene of mycobacteria, as verified by quantitative real-time PCR on a collection of mycobacteria and non-mycobacterial species. The real-time PCR method we developed was successfully used to detect mycobacteria in tap water and lake samples.
Conclusions
The results indicate that this real-time PCR method targeting the atpE gene can serve for highly specific detection and precise quantification of Mycobacterium spp. in environmental samples.
doi:10.1186/1471-2180-13-277
PMCID: PMC4219376  PMID: 24299240
Mycobacteria; atpE gene; Environmental samples
16.  The Critical Role of DNA Extraction for Detection of Mycobacteria in Tissues 
PLoS ONE  2013;8(10):e78749.
Background
Nucleic acid-based methods offer promise for both targeted and exploratory investigations of microbes in tissue samples. As the starting material for such studies is a mixture of host and microbial DNA, we have critically evaluated the DNA extraction step to determine the quantitative and qualitative parameters that permit faithful molecular detection of mycobacteria in infected tissue. Specifically, we assessed: 1) tissue disruption procedures; 2) DNA extraction protocols; and 3) inhibition of bacterial PCR by host DNA.
Principal Findings
Regarding DNA extraction, we found that 1) grinding was not necessary if bead-beating is done, 2) the reference mycobacterial DNA extraction method recovered more pure DNA than commercial spin column kits, 3) lysozyme digestion of 1 hour was sufficient, and 4) repeated steps of phenol:chloroform:isoamyl alcohol offered minimal gain in DNA quality. By artificially mixing mycobacterial DNA with DNA extracted from uninfected mice, we found that bacterial real-time quantitative PCR was only reliable when the quantity of host DNA was < 3 µg in a final volume of 25 µl and the quality was high (260/280 nm ratio = 1.89±0.08). Findings from spiked DNA studies were confirmed using DNA extracted from mice infected with different intracellular pathogens (M. tuberculosis, M. avium subsp. paratuberculosis).
Conclusions
Our findings point to the most appropriate methods for extracting DNA from tissue samples for the purpose of detecting and quantifying mycobacteria. These data also inform on the limits of detection for two mycobacterial species and indicate that increasing the sample mass to improve analytic sensitivity comes at the cost of inhibition of PCR by host DNA.
doi:10.1371/journal.pone.0078749
PMCID: PMC3806855  PMID: 24194951
17.  Reductions in intestinal Clostridiales precede the development of nosocomial Clostridium difficile infection 
Microbiome  2013;1:18.
Background
Antimicrobial use is thought to suppress the intestinal microbiota, thereby impairing colonization resistance and allowing Clostridium difficile to infect the gut. Additional risk factors such as proton-pump inhibitors may also alter the intestinal microbiota and predispose patients to Clostridium difficile infection (CDI). This comparative metagenomic study investigates the relationship between epidemiologic exposures, intestinal bacterial populations and subsequent development of CDI in hospitalized patients. We performed a nested case–control study including 25 CDI cases and 25 matched controls. Fecal specimens collected prior to disease onset were evaluated by 16S rRNA gene amplification and pyrosequencing to determine the composition of the intestinal microbiota during the at-risk period.
Results
The diversity of the intestinal microbiota was significantly reduced prior to an episode of CDI. Sequences corresponding to the phylum Bacteroidetes and to the families Bacteroidaceae and Clostridiales Incertae Sedis XI were depleted in CDI patients compared to controls, whereas sequences corresponding to the family Enterococcaceae were enriched. In multivariable analyses, cephalosporin and fluoroquinolone use, as well as a decrease in the abundance of Clostridiales Incertae Sedis XI were significantly and independently associated with CDI development.
Conclusions
This study shows that a reduction in the abundance of a specific bacterial family - Clostridiales Incertae Sedis XI - is associated with risk of nosocomial CDI and may represent a target for novel strategies to prevent this life-threatening infection.
doi:10.1186/2049-2618-1-18
PMCID: PMC3971611  PMID: 24450844
Intestinal microbiota; Clostridium difficile infection; 16S rRNA gene sequencing; Clostridiales Incertae Sedis XI
18.  Evaluation of a Semi-Automated Reporter Phage Assay for Susceptibility Testing of Mycobacterium tuberculosis Isolates in South Africa 
In a prospective study conducted by laboratory technologists in a diagnostic laboratory in Cape Town, South Africa, a semi-automated phage-based antibiotic susceptibility assay was implemented and the performance of the luciferase reporter mycobacteriophage (LRP) system for susceptibility testing of clinical Mycobacterium tuberculosis complex (MTC) isolates against rifampin and isoniazid was evaluated. Two hundred consecutive clinical MGIT cultures of MTC species were included in this study. Antibiotic susceptibility assays were setup manually for the LRP and BACTEC radiometric systems and read in a plate luminometer and the BACTEC 460 instrument, respectively. Discrepant susceptibility results were resolved by the conventional agar proportion method. Of the 200 secondary cultures prepared for this study, 9 (4.5%) were lost to contamination (LRP 4, BACTEC 1, both 4). All of the remaining 191 cultures underwent susceptibility testing by both methods and the overall agreement between the LRP and BACTEC was 98.4% (rifampin 100%; isoniazid 96.9%). Of the 6 discrepant cultures tested by the agar proportion method, 2 gave results in agreement with the LRP. The sensitivity of the LRP for detection of drug-resistant isolates was 100% for both rifampin (n=9) and isoniazid (n=12). The median turnaround time for susceptibility testing was 2 days with the LRP and 9 days with BACTEC. In conclusion, the semi-automated LRP-based assay offers a rapid and practical approach for accurate susceptibility testing of Mycobacterium tuberculosis cultures in diagnostic laboratories with limited financial resources but with competent technologists.
doi:10.1016/j.tube.2007.08.006
PMCID: PMC3677954  PMID: 17980664
Mycobacterium tuberculosis; drug susceptibility testing; luciferase reporter mycobacteriophages; drug resistance
19.  Vitamin D Induces Interleukin-1β Expression: Paracrine Macrophage Epithelial Signaling Controls M. tuberculosis Infection 
PLoS Pathogens  2013;9(6):e1003407.
Although vitamin D deficiency is a common feature among patients presenting with active tuberculosis, the full scope of vitamin D action during Mycobacterium tuberculosis (Mtb) infection is poorly understood. As macrophages are the primary site of Mtb infection and are sites of vitamin D signaling, we have used these cells to understand the molecular mechanisms underlying modulation of the immune response by the hormonal form of vitamin D, 1,25-dihydroxyvitamin D (1,25D). We found that the virulent Mtb strain H37Rv elicits a broad host transcriptional response. Transcriptome profiling also revealed that the profile of target genes regulated by 1,25D is substantially altered by infection, and that 1,25D generally boosts infection-stimulated cytokine/chemokine responses. We further focused on the role of 1,25D- and infection-induced interleukin 1β (IL-1β) expression in response to infection. 1,25D enhanced IL-1β expression via a direct transcriptional mechanism. Secretion of IL-1β from infected cells required the NLRP3/caspase-1 inflammasome. The impact of IL-1β production was investigated in a novel model wherein infected macrophages were co-cultured with primary human small airway epithelial cells. Co-culture significantly prolonged survival of infected macrophages, and 1,25D/infection-induced IL-1β secretion from macrophages reduced mycobacterial burden by stimulating the anti-mycobacterial capacity of co-cultured lung epithelial cells. These effects were independent of 1,25D-stimulated autophagy in macrophages but dependent upon epithelial IL1R1 signaling and IL-1β-driven epithelial production of the antimicrobial peptide DEFB4/HBD2. These data provide evidence that the anti-microbial actions of vitamin D extend beyond the macrophage by modulating paracrine signaling, reinforcing its role in innate immune regulation in humans.
Author Summary
In 2010 there were ∼9 million cases of tuberculosis and 1.4 million deaths, representing the second largest cause of death worldwide and the leading cause of death from a curable disease. M. tuberculosis (Mtb) replicates within cells of the immune system called macrophages over an approximate 72 hour period, ultimately inducing cell death. Notably, macrophages are sites of vitamin D signaling, and there is broad evidence that vitamin D modulates macrophage responses to Mtb. Elevated levels of TB have long been associated with vitamin D deficiency, strongly suggesting that vitamin D supplementation may be of therapeutic benefit. In this study we profile the host macrophage response to Mtb infection through the use of high-throughput genomics techniques. From this we have discovered that the dominant function of vitamin D is the modulation of the levels of specific cytokines, mediators of immune cell to cell signaling. Of particular interest was the increase in IL-1β signaling, which we show to be directly regulated by vitamin D. We also show that this increase in IL-1β is critical for driving a signaling cascade between macrophages and lung epithelial cells leading to epithelial antimicrobial peptide production that helps to contain Mtb infection in our model culture system.
doi:10.1371/journal.ppat.1003407
PMCID: PMC3675149  PMID: 23762029
20.  Repeat IGRA Testing in Canadian Health Workers: Conversions or Unexplained Variability? 
PLoS ONE  2013;8(1):e54748.
Background
Although North American hospitals are switching from tuberculin testing (TST) to interferon-gamma release assays (IGRAs), data are limited on the association between occupational exposure and serial QuantiFERON-TB Gold In-Tube (QFT) results in healthcare workers (HCWs).
Methods
In a cohort of Canadian HCWs, TST and QFT were performed at study enrolment (TST1 and QFT1) and 1 year later (TST2 and QFT2). Conversion and reversion rates were estimated, and correlation with TB exposure was assessed.
Results
Among 258 HCWs, median age was 36.8 years, 188/258 (73%) were female and 183/258 (71%) were Canadian-born. In 245 subjects with a negative QFT1 we found a QFT conversion rate of 5.3% (13/245, 95% CI 2.9–8.9%). Using more stringent definitions, QFT conversion rates ranged from 2.0 to 5.3%. No TST conversions were found among the 241 HCWs with negative TST1, and no measure of recent TB exposure was associated with QFT conversions. In the 13 HCWs with a positive QFT1, 62% reverted.
Conclusion
Using the conventional QFT conversion definition, we found a higher than expected rate of conversion. Recent occupational exposures were not associated with QFT conversions, and no TST conversions occurred in this cohort, suggesting the ‘conversions’ may not reflect new TB infection.
doi:10.1371/journal.pone.0054748
PMCID: PMC3561382  PMID: 23382955
21.  Mycobacterial disease and impaired IFN-γ immunity in humans with inherited ISG15 deficiency 
Science (New York, N.Y.)  2012;337(6102):1684-1688.
ISG15 is an interferon (IFN)-α/β-inducible, ubiquitin-like intracellular protein. Its conjugation to various proteins (ISGylation) contributes to antiviral immunity in mice. We describe human patients with inherited ISG15 deficiency and mycobacterial, but not viral diseases. The lack of intracellular ISG15 production and protein ISGylation was not associated with cellular susceptibility to any viruses tested, consistent with the lack of viral diseases in these patients. By contrast, the lack of mycobacterium-induced ISG15 secretion by leukocytes — granulocytes in particular — reduced the production of IFN-γ by lymphocytes, including natural killer cells, probably accounting for the enhanced susceptibility to mycobacterial disease. This experiment of Nature shows that human ISGylation is largely redundant for antiviral immunity, but that ISG15 plays an essential role as an IFN-γ-inducing secreted molecule for optimal antimycobacterial immunity.
doi:10.1126/science.1224026
PMCID: PMC3507439  PMID: 22859821
22.  Inter- and Intra-subtype genotypic differences that differentiate Mycobacterium avium subspecies paratuberculosis strains 
BMC Microbiology  2012;12:264.
Background
Mycobacterium avium subspecies paratuberculosis (Map) is the aetiological agent of Johne’s disease or paratuberculosis and is included within the Mycobacterium avium complex (MAC). Map strains are of two major types often referred to as ‘Sheep’ or ‘S-type’ and ‘Cattle’ or ‘C-type’. With the advent of more discriminatory typing techniques it has been possible to further classify the S-type strains into two groups referred to as Type I and Type III. This study was undertaken to genotype a large panel of S-type small ruminant isolates from different hosts and geographical origins and to compare them with a large panel of well documented C-type isolates to assess the genetic diversity of these strain types. Methods used included Mycobacterial Interspersed Repetitive Units - Variable-Number Tandem Repeat analysis (MIRU-VNTR), analysis of Large Sequence Polymorphisms by PCR (LSP analysis), Single Nucleotide Polymorphism (SNP) analysis of gyr genes, Pulsed-Field Gel Electrophoresis (PFGE) and Restriction Fragment Length Polymorphism analysis coupled with hybridization to IS900 (IS900-RFLP) analysis.
Results
The presence of LSPA4 and absence of LSPA20 was confirmed in all 24 Map S-type strains analysed. SNPs within the gyr genes divided the S-type strains into types I and III. Twenty four PFGE multiplex profiles and eleven different IS900-RFLP profiles were identified among the S-type isolates, some of them not previously published. Both PFGE and IS900-RFLP segregated the S-type strains into types I and III and the results concurred with those of the gyr SNP analysis. Nine MIRU-VNTR genotypes were identified in these isolates. MIRU-VNTR analysis differentiated Map strains from other members of Mycobacterium avium Complex, and Map S-type from C-type but not type I from III. Pigmented Map isolates were found of type I or III.
Conclusion
This is the largest panel of S-type strains investigated to date. The S-type strains could be further divided into two subtypes, I and III by some of the typing techniques (IS900-RFLP, PFGE and SNP analysis of the gyr genes). MIRU-VNTR did not divide the strains into the subtypes I and III but did detect genetic differences between isolates within each of the subtypes. Pigmentation is not exclusively associated with type I strains.
doi:10.1186/1471-2180-12-264
PMCID: PMC3546927  PMID: 23164429
23.  High yield of culture-based diagnosis in a TB-endemic setting 
BMC Infectious Diseases  2012;12:218.
Background
In most of the world, microbiologic diagnosis of tuberculosis (TB) is limited to microscopy. Recent guidelines recommend culture-based diagnosis where feasible.
Methods
In order to evaluate the relative and absolute incremental diagnostic yield of culture-based diagnosis in a high-incidence community in Cape Town, South Africa, subjects evaluated for suspected TB had their samples processed for microscopy and culture over a 21 month period.
Results
For 2537 suspect episodes with 2 smears and 2 cultures done, 20.0% (508) had at least one positive smear and 29.9% (760) had at least one positive culture. One culture yielded 1.8 times more cases as 1 smear (relative yield), or an increase of 12.0% (absolute yield). Based on the latter value, the number of cultures needed to diagnose (NND) one extra case of TB was 8, compared to 19 if second specimens were submitted for microscopy.
Conclusion
In a high-burden setting, the introduction of culture can markedly increase TB diagnosis over microscopy. The concept of number needed to diagnose can help in comparing incremental yield of diagnosis methods. Although new promising diagnostic molecular methods are being implemented, TB culture is still the gold standard.
doi:10.1186/1471-2334-12-218
PMCID: PMC3482573  PMID: 22978323
Tuberculosis; Diagnosis; Culture; Microscopy
24.  TB Screening in Canadian Health Care Workers Using Interferon-Gamma Release Assays 
PLoS ONE  2012;7(8):e43014.
Background
While many North American healthcare institutions are switching from Tuberculin Skin Test (TST) to Interferon-gamma release assays (IGRAs), there is relatively limited data on association between occupational tuberculosis (TB) risk factors and test positivity and/or patterns of test discordance.
Methods
We recruited a cohort of Canadian health care workers (HCWs) in Montreal, and performed both TST and QuantiFERON-TB Gold In Tube (QFT) tests, and assessed risk factors and occupational exposure.
Results
In a cross-sectional analysis of baseline results, the prevalence of TST positivity using the 10 mm cut-off was 5.7% (22/388, 95%CI: 3.6–8.5%), while QFT positivity was 6.2% (24/388, 95%CI: 4–9.1%). Overall agreement between the tests was poor (kappa = 0.26), and 8.3% of HCWs had discordant test results, most frequently TST−/QFT+ (17/388, 4.4%). TST positivity was associated with total years worked in health care, non-occupational exposure to TB and BCG vaccination received after infancy or on multiple occasions. QFT positivity was associated with having worked as a HCW in a foreign country.
Conclusions
Our results suggest that LTBI prevalence as measured by either the TST or the QFT is low in this HCW population. Of concern is the high frequency of unexplainable test discordance, namely: TST−/QFT+ subjects, and the lack of any association between QFT positivity and clear-cut recent TB exposure. If these discordant results are indeed false positives, the use of QFT in lieu of TST in low TB incidence settings could result in overtreatment of uninfected individuals.
doi:10.1371/journal.pone.0043014
PMCID: PMC3423433  PMID: 22916197
25.  An N-Ethyl-N-Nitrosourea (ENU)-Induced Dominant Negative Mutation in the JAK3 Kinase Protects against Cerebral Malaria 
PLoS ONE  2012;7(2):e31012.
Cerebral malaria (CM) is a lethal neurological complication of malaria. We implemented a genome-wide screen in mutagenized mice to identify host proteins involved in CM pathogenesis and whose inhibition may be of therapeutic value. One pedigree (P48) segregated a resistance trait whose CM-protective effect was fully penetrant, mapped to chromosome 8, and identified by genome sequencing as homozygosity for a mis-sense mutation (W81R) in the FERM domain of Janus-associated kinase 3 (Jak3). The causative effect of Jak3W81R was verified by complementation testing in Jak3W81R/− double heterozygotes that were fully protected against CM. Jak3W81R homozygotes showed defects in thymic development with depletion of CD8+ T cell, B cell, and NK cell compartments, and defective T cell-dependent production of IFN-γ. Adoptive transfer of normal splenocytes abrogates CM resistance in Jak3W81R homozygotes, an effect attributed to the CD8+ T cells. Jak3W81R behaves as a dominant negative variant, with significant CM resistance of Jak3W81R/+ heterozygotes, compared to CM-susceptible Jak3+/+ and Jak3+/− controls. CM resistance in Jak3W81R/+ heterozygotes occurs in presence of normal T, B and NK cell numbers. These findings highlight the pathological role of CD8+ T cells and Jak3-dependent IFN-γ-mediated Th1 responses in CM pathogenesis.
doi:10.1371/journal.pone.0031012
PMCID: PMC3283600  PMID: 22363534

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