Publications by authors named "Mireia Coscolla"

45 Publications

Local adaptation in populations of endemic to the Indian Ocean Rim.

F1000Res 2021 1;10:60. Epub 2021 Feb 1.

Institut Pasteur de Madagascar, Antananarivo, Madagascar.

Lineage 1 (L1) and 3 (L3) are two lineages of the complex (MTBC) causing tuberculosis (TB) in humans. L1 and L3 are prevalent around the rim of the Indian Ocean, the region that accounts for most of the world's new TB cases. Despite their relevance for this region, L1 and L3 remain understudied. We analyzed 2,938 L1 and 2,030 L3 whole genome sequences originating from 69 countries. We reconstructed the evolutionary history of these two lineages and identified genes under positive selection. We found a strongly asymmetric pattern of migration from South Asia toward neighboring regions, highlighting the historical role of South Asia in the dispersion of L1 and L3. Moreover, we found that several genes were under positive selection, including genes involved in virulence and resistance to antibiotics. For L1 we identified signatures of local adaptation at the locus, a gene coding for a secreted effector that targets the human endosomal sorting complex, and is included in several vaccine candidates. Our study highlights the importance of genetic diversity in the MTBC, and sheds new light on two of the most important MTBC lineages affecting humans.
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http://dx.doi.org/10.12688/f1000research.28318.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7921886.2PMC
February 2021

Phylogenomics of reveals a new lineage and a complex evolutionary history.

Microb Genom 2021 02;7(2)

University of Basel, Basel, Switzerland.

Human tuberculosis (TB) is caused by members of the complex (MTBC). The MTBC comprises several human-adapted lineages known as , as well as two lineages (L5 and L6) traditionally referred to as . Strains of L5 and L6 are largely limited to West Africa for reasons unknown, and little is known of their genomic diversity, phylogeography and evolution. Here, we analysed the genomes of 350 L5 and 320 L6 strains, isolated from patients from 21 African countries, plus 5 related genomes that had not been classified into any of the known MTBC lineages. Our population genomic and phylogeographical analyses showed that the unclassified genomes belonged to a new group that we propose to name MTBC lineage 9 (L9). While the most likely ancestral distribution of L9 was predicted to be East Africa, the most likely ancestral distribution for both L5 and L6 was the Eastern part of West Africa. Moreover, we found important differences between L5 and L6 strains with respect to their phylogeographical substructure and genetic diversity. Finally, we could not confirm the previous association of drug-resistance markers with lineage and sublineages. Instead, our results indicate that the association of drug resistance with lineage is most likely driven by sample bias or geography. In conclusion, our study sheds new light onto the genomic diversity and evolutionary history of , and highlights the need to consider the particularities of each MTBC lineage for understanding the ecology and epidemiology of TB in Africa and globally.
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http://dx.doi.org/10.1099/mgen.0.000477DOI Listing
February 2021

Reference set of Mycobacterium tuberculosis clinical strains: A tool for research and product development.

PLoS One 2019 25;14(3):e0214088. Epub 2019 Mar 25.

Swiss Tropical and Public Health Institute, Basel, Switzerland.

The Mycobacterium tuberculosis complex (MTBC) causes tuberculosis (TB) in humans and various other mammals. The human-adapted members of the MTBC comprise seven phylogenetic lineages that differ in their geographical distribution. There is growing evidence that this phylogeographic diversity modulates the outcome of TB infection and disease. For decades, TB research and development has focused on the two canonical MTBC laboratory strains H37Rv and Erdman, both of which belong to Lineage 4. Relying on only a few laboratory-adapted strains can be misleading as study results might not be directly transferrable to clinical settings where patients are infected with a diverse array of strains, including drug-resistant variants. Here, we argue for the need to expand TB research and development by incorporating the phylogenetic diversity of the MTBC. To facilitate such work, we have assembled a group of 20 genetically well-characterized clinical strains representing the seven known human-adapted MTBC lineages. With the "MTBC clinical strains reference set" we aim to provide a standardized resource for the TB community. We hope it will enable more direct comparisons between studies that explore the physiology of MTBC beyond the laboratory strains used thus far. We anticipate that detailed phenotypic analyses of this reference strain set will increase our understanding of TB biology and assist in the development of new control tools that are broadly effective.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0214088PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6433267PMC
December 2019

Molecular epidemiology and whole genome sequencing analysis of clinical Mycobacterium bovis from Ghana.

PLoS One 2019 4;14(3):e0209395. Epub 2019 Mar 4.

Noguchi Memorial Institute for Medical Research, College of Health Sciences, University of Ghana, Legon, Accra, Ghana.

Background: Bovine tuberculosis (bTB) caused by Mycobacterium bovis is a re-emerging problem in both livestock and humans. The association of some M. bovis strains with hyper-virulence, MDR-TB and disseminated disease makes it imperative to understand the biology of the pathogen.

Methods: Mycobacterium bovis (15) among 1755 M. tuberculosis complex (MTBC) isolated between 2012 and 2014 were characterized and analyzed for associated patient demography and other risk factors. Five of the M. bovis isolates were whole-genome sequenced and comparatively analyzed against a global collection of published M. bovis genomes.

Results: Mycobacterium bovis was isolated from 3/560(0.5%) females and 12/1195(1.0%) males with pulmonary TB. The average age of M. bovis infected cases was 46.8 years (7-72years). TB patients from the Northern region of Ghana (1.9%;4/212) had a higher rate of infection with M. bovis (OR = 2.7,p = 0.0968) compared to those from the Greater Accra region (0.7%;11/1543). Among TB patients with available HIV status, the odds of isolating M. bovis from HIV patients (2/119) was 3.3 higher relative to non-HIV patients (4/774). Direct contact with livestock or their unpasteurized products was significantly associated with bTB (p<0.0001, OR = 124.4,95% CI = 30.1-508.3). Two (13.3%) of the M. bovis isolates were INH resistant due to the S315T mutation in katG whereas one (6.7%) was RIF resistant with Q432P and I1491S mutations in rpoB. M. bovis from Ghana resolved as mono-phyletic branch among mostly M. bovis from Africa irrespective of the host and were closest to the root of the global M. bovis phylogeny. M. bovis-specific amino acid mutations were detected among MTBC core genes such as mce1A, mmpL1, pks6, phoT, pstB, glgP and Rv2955c. Additional mutations P6T in chaA, G187E in mgtC, T35A in Rv1979c, S387A in narK1, L400F in fas and A563T in eccA1 were restricted to the 5 clinical M. bovis from Ghana.

Conclusion: Our data indicate potential zoonotic transmission of bTB in Ghana and hence calls for intensified public education on bTB, especially among risk groups.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0209395PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6398925PMC
November 2019

A New Phylogenetic Framework for the Animal-Adapted Complex.

Front Microbiol 2018 27;9:2820. Epub 2018 Nov 27.

Swiss Tropical and Public Health Institute, Basel, Switzerland.

Tuberculosis (TB) affects humans and other animals and is caused by bacteria from the complex (MTBC). Previous studies have shown that there are at least nine members of the MTBC infecting animals other than humans; these have also been referred to as ecotypes. However, the ecology and the evolution of these animal-adapted MTBC ecotypes are poorly understood. Here we screened 12,886 publicly available MTBC genomes and newly sequenced 17 animal-adapted MTBC strains, gathering a total of 529 genomes of animal-adapted MTBC strains. Phylogenomic and comparative analyses confirm that the animal-adapted MTBC members are paraphyletic with some members more closely related to the human-adapted Lineage 6 than to other animal-adapted strains. Furthermore, we identified four main animal-adapted MTBC clades that might correspond to four main host shifts; two of these clades are hypothesized to reflect independent cattle domestication events. Contrary to what would be expected from an obligate pathogen, MTBC nucleotide diversity was not positively correlated with host phylogenetic distances, suggesting that host tropism in the animal-adapted MTBC seems to be driven by contact rates and demographic aspects of the host population rather by than host relatedness. By combining phylogenomics with ecological data, we propose an evolutionary scenario in which the ancestor of Lineage 6 and all animal-adapted MTBC ecotypes was a generalist pathogen that subsequently adapted to different host species. This study provides a new phylogenetic framework to better understand the evolution of the different ecotypes of the MTBC and guide future work aimed at elucidating the molecular mechanisms underlying host range.
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http://dx.doi.org/10.3389/fmicb.2018.02820DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6277475PMC
November 2018

Low sensitivity of the MPT64 identification test to detect lineage 5 of the Mycobacterium tuberculosis complex.

J Med Microbiol 2018 Dec 2;67(12):1718-1727. Epub 2018 Nov 2.

2​Laboratoire de Référence des Mycobactéries, Cotonou, Benin.

Purpose: Differentiation of the Mycobacterium tuberculosis complex (MTBc) from non-tuberculous mycobacteria (NTM) is important for tuberculosis diagnosis and is a prerequisite for reliable phenotypic drug-resistance testing. We evaluated the performance of the rapid MPT64 antigen identification test for the detection of Mycobacterium africanum lineage 5 (MAF L5).

Methodology: Smear-positive tuberculosis patients' sputa were included prospectively. Culture was performed on Löwenstein-Jensen medium and, when positive, the MPT64 test and the classical para-nitro benzoic acid susceptibility and heat-labile catalase (PNB/catalase) identification tests were performed. The MPT64 test was repeated 14 days after an initially negative first testing. Direct spoligotyping was performed for MTBc lineage determination.

Results: In total, 333 isolates were tested for all of the methods. Three hundred and twenty-two (96.7 %) were pure MTBc, by agreement between spoligotyping and PNB/catalase, and 11 were NTM or a mixture of MTBc/NTM. The MPT64 test conducted on day zero of culture-positivity correctly identified most of the pure MTBc isolates (93.2 %, 300/322), but it failed to detect 24 % of the L5 isolates (18/75) versus 2 % (4/202) of the L4 ones [OR=15.6 (5.3-45.8), P<0.0001], with improved sensitivity for L5 detection on repeat testing after 14 days. The L5-wide non-synonymous single-nucleotide polymorphism in the mpt64 gene may explain the poor performance of the MPT64 test for L5.

Conclusion: The MPT64 test has a lower sensitivity for detecting L5 isolates of the MTBc, and can be considered as a first-screening test that should be confirmed by another identification method when it produces negative results in countries with L5. Given the microbiological bias in both the isolation and identification of MAF lineages, diagnostics with high sensitivity for direct testing on clinical material are preferable.
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http://dx.doi.org/10.1099/jmm.0.000846DOI Listing
December 2018

Comparative genomics of Mycobacterium africanum Lineage 5 and Lineage 6 from Ghana suggests distinct ecological niches.

Sci Rep 2018 07 26;8(1):11269. Epub 2018 Jul 26.

Noguchi Memorial Institute for Medical Research, University of Ghana, Legon, Accra, Ghana.

Mycobacterium africanum (Maf) causes a substantial proportion of human tuberculosis in some countries of West Africa, but little is known on this pathogen. We compared the genomes of 253 Maf clinical isolates from Ghana, including N = 175 Lineage 5 (L5) and N = 78 Lineage 6 (L6). We found that the genomic diversity of L6 was higher than in L5 despite the smaller sample size. Regulatory proteins appeared to evolve neutrally in L5 but under purifying selection in L6. Even though over 90% of the human T cell epitopes were conserved in both lineages, L6 showed a higher ratio of non-synonymous to synonymous single nucleotide variation in these epitopes overall compared to L5. Of the 10% human T cell epitopes that were variable, most carried mutations that were lineage-specific. Our findings indicate that Maf L5 and L6 differ in some of their population genomic characteristics, possibly reflecting different selection pressures linked to distinct ecological niches.
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http://dx.doi.org/10.1038/s41598-018-29620-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6062541PMC
July 2018

Tuberculosis outbreak investigation using phylodynamic analysis.

Epidemics 2018 12 22;25:47-53. Epub 2018 May 22.

Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland; Swiss Institute of Bioinformatics (SIB), Lausanne, Switzerland.

The fast evolution of pathogenic viruses has allowed for the development of phylodynamic approaches that extract information about the epidemiological characteristics of viral genomes. Thanks to advances in whole genome sequencing, they can be applied to slowly evolving bacterial pathogens like Mycobacterium tuberculosis. In this study, we investigate and compare the epidemiological dynamics underlying two M. tuberculosis outbreaks using phylodynamic methods. Specifically, we (i) test if the outbreak data sets contain enough genetic variation to estimate short-term evolutionary rates and (ii) reconstruct epidemiological parameters such as the effective reproduction number. The first outbreak occurred in the Swiss city of Bern (1987-2012) and was caused by a drug-susceptible strain belonging to the phylogenetic M. tuberculosis Lineage 4. The second outbreak was caused by a multidrug-resistant (MDR) strain of Lineage 2, imported from the Wat Tham Krabok (WTK) refugee camp in Thailand into California. There is little temporal signal in the Bern data set and moderate temporal signal in the WTK data set. Thanks to its high sampling proportion (90%) the Bern outbreak allows robust estimation of epidemiological parameters despite the poor temporal signal. Conversely, there is much uncertainty in the epidemiological estimates concerning the sparsely sampled (9%) WTK outbreak. Our results suggest that both outbreaks peaked around 1990, although they were only recognized as outbreaks in 1993 (Bern) and 2004 (WTK). Furthermore, individuals were infected for a significantly longer period (around 9 years) in the WTK outbreak than in the Bern outbreak (4-5 years). Our work highlights both the limitations and opportunities of phylodynamic analysis of outbreaks involving slowly evolving pathogens: (i) estimation of the evolutionary rate is difficult on outbreak time scales and (ii) a high sampling proportion allows quantification of the age of the outbreak based on the sampling times, and thus allows for robust estimation of epidemiological parameters.
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http://dx.doi.org/10.1016/j.epidem.2018.05.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6227250PMC
December 2018

Treemmer: a tool to reduce large phylogenetic datasets with minimal loss of diversity.

BMC Bioinformatics 2018 05 2;19(1):164. Epub 2018 May 2.

Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, Basel, Switzerland.

Background: Large sequence datasets are difficult to visualize and handle. Additionally, they often do not represent a random subset of the natural diversity, but the result of uncoordinated and convenience sampling. Consequently, they can suffer from redundancy and sampling biases.

Results: Here we present Treemmer, a simple tool to evaluate the redundancy of phylogenetic trees and reduce their complexity by eliminating leaves that contribute the least to the tree diversity.

Conclusions: Treemmer can reduce the size of datasets with different phylogenetic structures and levels of redundancy while maintaining a sub-sample that is representative of the original diversity. Additionally, it is possible to fine-tune the behavior of Treemmer including any kind of meta-information, making Treemmer particularly useful for empirical studies.
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http://dx.doi.org/10.1186/s12859-018-2164-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5930393PMC
May 2018

Biological and Epidemiological Consequences of MTBC Diversity.

Authors:
Mireia Coscolla

Adv Exp Med Biol 2017 ;1019:95-116

Swiss Tropical and Public Health Institute, University of Basel, Socinstrasse 57, 4002, Basel, Switzerland.

Tuberculosis is caused by different groups of bacteria belonging to the Mycobacterium tuberculosis complex (MTBC). The combined action of human factors, environmental conditions and bacterial virulence determine the extent and form of human disease. MTBC virulence is a composite of different clinical phenotypes such as transmission rate and disease severity among others. Clinical phenotypes are also influenced by cellular and immunological phenotypes. MTBC phenotypes are determined by the genotype, therefore finding genotypes responsible for clinical phenotypes would allow discovering MTBC virulence factors. Different MTBC strains display different cellular and clinical phenotypes. Strains from Lineage 5 and Lineage 6 are metabolically different, grow slower, and are less virulent. Also, at least certain groups of Lineage 2 and Lineage 4 strains are more virulent in terms of disease severity and human-to-human transmission. Because phenotypic differences are ultimately caused by genotypic differences, different genomic loci have been related to various cellular and clinical phenotypes. However, defining the impact of specific bacterial genomic loci on virulence when other bacterial determinants, human and environmental factors are also impacting the phenotype would contribute to a better knowledge of tuberculosis virulence and ultimately benefit tuberculosis control.
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http://dx.doi.org/10.1007/978-3-319-64371-7_5DOI Listing
March 2018

Tuberculosis in Swiss captive Asian elephants: microevolution of Mycobacterium tuberculosis characterized by multilocus variable-number tandem-repeat analysis and whole-genome sequencing.

Sci Rep 2017 11 7;7(1):14647. Epub 2017 Nov 7.

Swiss Tropical and Public Health Institute, CH-4002, Basel, Switzerland.

Zoonotic tuberculosis is a risk for human health, especially when animals are in close contact with humans. Mycobacterium tuberculosis was cultured from several organs, including lung tissue and gastric mucosa, of three captive elephants euthanized in a Swiss zoo. The elephants presented weight loss, weakness and exercise intolerance. Molecular characterization of the M. tuberculosis isolates by spoligotyping revealed an identical profile, suggesting a single source of infection. Multilocus variable-number of tandem-repeat analysis (MLVA) elucidated two divergent populations of bacteria and mixed infection in one elephant, suggesting either different transmission chains or prolonged infection over time. A total of eight M. tuberculosis isolates were subjected to whole-genome sequence (WGS) analysis, confirming a single source of infection and indicating the route of transmission between the three animals. Our findings also show that the methods currently used for epidemiological investigations of M. tuberculosis infections should be carefully applied on isolates from elephants. Moreover the importance of multiple sampling and analysis of within-host mycobacterial clonal populations for investigations of transmission is demonstrated.
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http://dx.doi.org/10.1038/s41598-017-15278-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5676744PMC
November 2017

Significant under expression of the DosR regulon in M. tuberculosis complex lineage 6 in sputum.

Tuberculosis (Edinb) 2017 05 4;104:58-64. Epub 2017 Mar 4.

Mycobacteriology Unit, Institute of Tropical Medicine (ITM), Nationalestraat 155, 2000, Antwerp, Belgium. Electronic address:

Mycobacterium africanum lineage (L) 6 is an important pathogen in West Africa, causing up to 40% of pulmonary tuberculosis (TB). The biology underlying the clinical differences between M. africanum and M. tuberculosis sensu stricto remains poorly understood. We performed ex vivo expression of 2179 genes of the most geographically dispersed cause of human TB, M. tuberculosis L4 and the geographically restricted, M. africanum L6 directly from sputa of 11 HIV-negative TB patients from The Gambia who had not started treatment. The DosR regulon was the most significantly decreased category in L6 relative to L4. Further, we identified nonsynonymous mutations in major DosR regulon genes of 44 L6 genomes of TB patients from The Gambia and Ghana. Using Lebek's test, we assessed differences in oxygen requirements for growth. L4 grew only at the aerobic surface while L6 grew throughout the medium. In the host, the DosR regulon is critical for M. tuberculosis in adaptation to oxygen limitation. However, M. africanum L6 appears to have adapted to growth under hypoxic conditions or to different biological niches. The observed under expression of DosR in L6 fits with the genomic changes in DosR genes, microaerobic growth and the association with extrapulmonary disease.
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http://dx.doi.org/10.1016/j.tube.2017.03.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5421582PMC
May 2017

Reversion of antibiotic resistance in by spiroisoxazoline SMARt-420.

Science 2017 Mar 16;355(6330):1206-1211. Epub 2017 Mar 16.

Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019-UMR8204-CIIL-Center for Infection and Immunity of Lille, F-59000 Lille, France.

Antibiotic resistance is one of the biggest threats to human health globally. Alarmingly, multidrug-resistant and extensively drug-resistant have now spread worldwide. Some key antituberculosis antibiotics are prodrugs, for which resistance mechanisms are mainly driven by mutations in the bacterial enzymatic pathway required for their bioactivation. We have developed drug-like molecules that activate a cryptic alternative bioactivation pathway of ethionamide in , circumventing the classic activation pathway in which resistance mutations have now been observed. The first-of-its-kind molecule, named SMARt-420 (Small Molecule Aborting Resistance), not only fully reverses ethionamide-acquired resistance and clears ethionamide-resistant infection in mice, it also increases the basal sensitivity of bacteria to ethionamide.
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http://dx.doi.org/10.1126/science.aag1006DOI Listing
March 2017

Within Host Evolution Selects for a Dominant Genotype of Mycobacterium tuberculosis while T Cells Increase Pathogen Genetic Diversity.

PLoS Pathog 2016 Dec 14;12(12):e1006111. Epub 2016 Dec 14.

Division of Infectious Diseases, Department of Medicine, New York University School of Medicine, New York, NY, United States of America.

Molecular epidemiological assessments, drug treatment optimization, and development of immunological interventions all depend on understanding pathogen adaptation and genetic variation, which differ for specific pathogens. Mycobacterium tuberculosis is an exceptionally successful human pathogen, yet beyond knowledge that this bacterium has low overall genomic variation but acquires drug resistance mutations, little is known of the factors that drive its population genomic characteristics. Here, we compared the genetic diversity of the bacteria that established infection to the bacterial populations obtained from infected tissues during murine M. tuberculosis pulmonary infection and human disseminated M. bovis BCG infection. We found that new mutations accumulate during in vitro culture, but that in vivo, purifying selection against new mutations dominates, indicating that M. tuberculosis follows a dominant lineage model of evolution. Comparing bacterial populations passaged in T cell-deficient and immunocompetent mice, we found that the presence of T cells is associated with an increase in the diversity of the M. tuberculosis genome. Together, our findings put M. tuberculosis genetic evolution in a new perspective and clarify the impact of T cells on sequence diversity of M. tuberculosis.
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http://dx.doi.org/10.1371/journal.ppat.1006111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5189959PMC
December 2016

Mycobacterium tuberculosis lineage 4 comprises globally distributed and geographically restricted sublineages.

Nat Genet 2016 12 31;48(12):1535-1543. Epub 2016 Oct 31.

LabPlus, Auckland City Hospital, Auckland, New Zealand.

Generalist and specialist species differ in the breadth of their ecological niches. Little is known about the niche width of obligate human pathogens. Here we analyzed a global collection of Mycobacterium tuberculosis lineage 4 clinical isolates, the most geographically widespread cause of human tuberculosis. We show that lineage 4 comprises globally distributed and geographically restricted sublineages, suggesting a distinction between generalists and specialists. Population genomic analyses showed that, whereas the majority of human T cell epitopes were conserved in all sublineages, the proportion of variable epitopes was higher in generalists. Our data further support a European origin for the most common generalist sublineage. Hence, the global success of lineage 4 reflects distinct strategies adopted by different sublineages and the influence of human migration.
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http://dx.doi.org/10.1038/ng.3704DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5238942PMC
December 2016

Standard Genotyping Overestimates Transmission of Mycobacterium tuberculosis among Immigrants in a Low-Incidence Country.

J Clin Microbiol 2016 07 18;54(7):1862-1870. Epub 2016 May 18.

Swiss Tropical and Public Health Institute, Basel, Switzerland

Immigrants from regions with a high incidence of tuberculosis (TB) are a risk group for TB in low-incidence countries such as Switzerland. In a previous analysis of a nationwide collection of 520 Mycobacterium tuberculosis isolates from 2000 to 2008, we identified 35 clusters comprising 90 patients based on standard genotyping (24-locus mycobacterial interspersed repetitive-unit-variable-number tandem-repeat [MIRU-VNTR] typing and spoligotyping). Here, we used whole-genome sequencing (WGS) to revisit these transmission clusters. Genome-based transmission clusters were defined as isolate pairs separated by ≤12 single nucleotide polymorphisms (SNPs). WGS confirmed 17/35 (49%) MIRU-VNTR typing clusters; the other 18 clusters contained pairs separated by >12 SNPs. Most transmission clusters (3/4) of Swiss-born patients were confirmed by WGS, as opposed to 25% (4/16) of the clusters involving only foreign-born patients. The overall clustering proportion was 17% (90 patients; 95% confidence interval [CI], 14 to 21%) by standard genotyping but only 8% (43 patients; 95% CI, 6 to 11%) by WGS. The clustering proportion was 17% (67/401; 95% CI, 13 to 21%) by standard genotyping and 7% (26/401; 95% CI, 4 to 9%) by WGS among foreign-born patients and 19% (23/119; 95% CI, 13 to 28%) and 14% (17/119; 95% CI, 9 to 22%), respectively, among Swiss-born patients. Using weighted logistic regression, we found weak evidence of an association between birth origin and transmission (adjusted odds ratio of 2.2 and 95% CI of 0.9 to 5.5 comparing Swiss-born patients to others). In conclusion, standard genotyping overestimated recent TB transmission in Switzerland compared to WGS, particularly among immigrants from regions with a high TB incidence, where genetically closely related strains often predominate. We recommend the use of WGS to identify transmission clusters in settings with a low incidence of TB.
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http://dx.doi.org/10.1128/JCM.00126-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4922098PMC
July 2016

M. tuberculosis T Cell Epitope Analysis Reveals Paucity of Antigenic Variation and Identifies Rare Variable TB Antigens.

Cell Host Microbe 2015 Nov;18(5):538-48

Pathogens that evade adaptive immunity typically exhibit antigenic variation. By contrast, it appears that although the chronic human tuberculosis (TB)-causing pathogen Mycobacterium tuberculosis needs to counter host T cell responses, its T cell epitopes are hyperconserved. Here we present an extensive analysis of the T cell epitopes of M. tuberculosis. We combined population genomics with experimental immunology to determine the number and identity of T cell epitope sequence variants in 216 phylogenetically diverse strains of M. tuberculosis. Antigen conservation is indeed a hallmark of M. tuberculosis. However, our analysis revealed a set of seven variable antigens that were immunogenic in subjects with active TB. These findings suggest that M. tuberculosis uses mechanisms other than antigenic variation to evade T cells. T cell epitopes that exhibit sequence variation may not be subject to the same evasion mechanisms, and hence vaccines that include such variable epitopes may be more efficacious.
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http://dx.doi.org/10.1016/j.chom.2015.10.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4758912PMC
November 2015

Acquired Resistance to Bedaquiline and Delamanid in Therapy for Tuberculosis.

N Engl J Med 2015 Nov;373(20):1986-8

University of Zurich, Zurich, Switzerland.

Treatment of multidrug-resistant Mycobacterium tuberculosis is a challenge. This letter describes the emergence of resistance to new therapies, bedaquiline and delamanid.
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http://dx.doi.org/10.1056/NEJMc1505196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4681277PMC
November 2015

Geographical and Temporal Structures of Legionella pneumophila Sequence Types in Comunitat Valenciana (Spain), 1998 to 2013.

Appl Environ Microbiol 2015 Oct 31;81(20):7106-13. Epub 2015 Jul 31.

Joint Research Unit Infección y Salud, FISABIO-Universidad de Valencia, Instituto Cavanilles de Biodiversidad y Biología Evolutiva, Valencia, Spain CIBER en Epidemiología y Salud Pública, Valencia, Spain

Legionella pneumophila is an accidental human pathogen associated with aerosol formation in water-related sources. High recombination rates make Legionella populations genetically diverse, and nearly 2,000 different sequence types (STs) have been described to date for this environmental pathogen. The spatial distribution of STs is extremely heterogeneous, with some variants being present worldwide and others being detected at only a local scale. Similarly, some STs have been associated with disease outbreaks, such as ST578 or ST23. Spain is among the European countries with the highest incidences of reported legionellosis cases, and specifically, Comunitat Valenciana (CV) is the second most affected area in the country. In this work, we aimed at studying the overall diversity of Legionella pneumophila populations found in the period from 1998 to 2013 in 79 localities encompassing 23 regions within CV. To do so, we performed sequence-based typing (SBT) on 1,088 L. pneumophila strains detected in the area from both environmental and clinical sources. A comparison with the genetic structuring detected in a global data set that included 20 European and 7 non-European countries was performed. Our results reveal a level of diversity in CV that can be considered representative of the diversity found in other countries worldwide.
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http://dx.doi.org/10.1128/AEM.02196-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4579446PMC
October 2015

In vivo biosynthesis of terpene nucleosides provides unique chemical markers of Mycobacterium tuberculosis infection.

Chem Biol 2015 Apr;22(4):516-526

Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Harvard Medical School, Smith Building Room 538, 1 Jimmy Fund Way, Boston, MA 02115, USA. Electronic address:

Although small molecules shed from pathogens are widely used to diagnose infection, such tests have not been widely implemented for tuberculosis. Here we show that the recently identified compound, 1-tuberculosinyladenosine (1-TbAd), accumulates to comprise >1% of all Mycobacterium tuberculosis lipid. In vitro and in vivo, two isomers of TbAd were detected that might serve as infection markers. Using mass spectrometry and nuclear magnetic resonance, we established the structure of the previously unknown molecule, N(6)-tuberculosinyladenosine (N(6)-TbAd). Its biosynthesis involves enzymatic production of 1-TbAd by Rv3378c followed by conversion to N(6)-TbAd via the Dimroth rearrangement. Intact biosynthetic genes are observed only within M. tuberculosis complex bacteria, and TbAd was not detected among other medically important pathogens, environmental bacteria, and vaccine strains. With no substantially similar known molecules in nature, the discovery and in vivo detection of two abundant terpene nucleosides support their development as specific diagnostic markers of tuberculosis.
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http://dx.doi.org/10.1016/j.chembiol.2015.03.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4432472PMC
April 2015

DNA replication fidelity in Mycobacterium tuberculosis is mediated by an ancestral prokaryotic proofreader.

Nat Genet 2015 Jun 20;47(6):677-81. Epub 2015 Apr 20.

Medical Research Council (MRC) Laboratory of Molecular Biology, Cambridge, UK.

The DNA replication machinery is an important target for antibiotic development in increasingly drug-resistant bacteria, including Mycobacterium tuberculosis. Although blocking DNA replication leads to cell death, disrupting the processes used to ensure replication fidelity can accelerate mutation and the evolution of drug resistance. In Escherichia coli, the proofreading subunit of the replisome, the ɛ exonuclease, is essential for high-fidelity DNA replication; however, we find that the corresponding subunit is completely dispensable in M. tuberculosis. Rather, the mycobacterial replicative polymerase DnaE1 itself encodes an editing function that proofreads DNA replication, mediated by an intrinsic 3'-5' exonuclease activity within its PHP domain. Inactivation of the DnaE1 PHP domain increases the mutation rate by more than 3,000-fold. Moreover, phylogenetic analysis of DNA replication proofreading in the bacterial kingdom suggests that E. coli is a phylogenetic outlier and that PHP domain-mediated proofreading is widely conserved and indeed may be the ancestral prokaryotic proofreader.
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http://dx.doi.org/10.1038/ng.3269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4449270PMC
June 2015

Genomic epidemiology of multidrug-resistant Mycobacterium tuberculosis during transcontinental spread.

J Infect Dis 2015 Jul 18;212(2):302-10. Epub 2015 Jan 18.

Division of Pulmonary and Critical Care Medicine, Francis J. Curry International Tuberculosis Center, San Francisco General Hospital, University of California.

The transcontinental spread of multidrug-resistant (MDR) tuberculosis is poorly characterized in molecular epidemiologic studies. We used genomic sequencing to understand the establishment and dispersion of MDR Mycobacterium tuberculosis within a group of immigrants to the United States. We used a genomic epidemiology approach to study a genotypically matched (by spoligotype, IS6110 restriction fragment length polymorphism, and mycobacterial interspersed repetitive units-variable number of tandem repeat signature) lineage 2/Beijing MDR strain implicated in an outbreak of tuberculosis among refugees in Thailand and consecutive cases within California. All 46 MDR M. tuberculosis genomes from both Thailand and California were highly related, with a median difference of 10 single-nucleotide polymorphisms (SNPs). The Wat Tham Krabok (WTK) strain is a new sequence type distinguished from all known Beijing strains by 55 SNPs and a genomic deletion (Rv1267c) associated with increased fitness. Sequence data revealed a highly prevalent MDR strain that included several closely related but distinct allelic variants within Thailand, rather than the occurrence of a single outbreak. In California, sequencing data supported multiple independent introductions of WTK with subsequent transmission and reactivation within the state, as well as a potential super spreader with a prolonged infectious period. Twenty-seven drug resistance-conferring mutations and 4 putative compensatory mutations were found within WTK strains. Genomic sequencing has substantial epidemiologic value in both low- and high-burden settings in understanding transmission chains of highly prevalent MDR strains.
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http://dx.doi.org/10.1093/infdis/jiv025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4490235PMC
July 2015

Consequences of genomic diversity in Mycobacterium tuberculosis.

Semin Immunol 2014 Dec 22;26(6):431-44. Epub 2014 Oct 22.

Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, Socinstrasse 57, 4002 Basel, Switzerland; University of Basel, Petersplatz 1, Basel 4003, Switzerland. Electronic address:

The causative agent of human tuberculosis, Mycobacterium tuberculosis complex (MTBC), comprises seven phylogenetically distinct lineages associated with different geographical regions. Here we review the latest findings on the nature and amount of genomic diversity within and between MTBC lineages. We then review recent evidence for the effect of this genomic diversity on mycobacterial phenotypes measured experimentally and in clinical settings. We conclude that overall, the most geographically widespread Lineage 2 (includes Beijing) and Lineage 4 (also known as Euro-American) are more virulent than other lineages that are more geographically restricted. This increased virulence is associated with delayed or reduced pro-inflammatory host immune responses, greater severity of disease, and enhanced transmission. Future work should focus on the interaction between MTBC and human genetic diversity, as well as on the environmental factors that modulate these interactions.
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http://dx.doi.org/10.1016/j.smim.2014.09.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4314449PMC
December 2014

Tracking a tuberculosis outbreak over 21 years: strain-specific single-nucleotide polymorphism typing combined with targeted whole-genome sequencing.

J Infect Dis 2015 Apr 30;211(8):1306-16. Epub 2014 Oct 30.

Swiss Tropical and Public Health Institute University of Basel Institute of Social and Preventive Medicine.

Background: Whole-genome sequencing (WGS) is increasingly used in molecular-epidemiological investigations of bacterial pathogens, despite cost- and time-intensive analyses. We combined strain-specific single-nucleotide polymorphism (SNP) typing and targeted WGS to investigate a tuberculosis cluster spanning 21 years in Bern, Switzerland.

Methods: On the basis of genome sequences of 3 historical outbreak Mycobacterium tuberculosis isolates, we developed a strain-specific SNP-typing assay to identify further cases. We screened 1642 patient isolates and performed WGS on all identified cluster isolates. We extracted SNPs to construct genomic networks. Clinical and social data were retrospectively collected.

Results: We identified 68 patients associated with the outbreak strain. Most received a tuberculosis diagnosis in 1991-1995, but cases were observed until 2011. Two thirds were homeless and/or substance abusers. Targeted WGS revealed 133 variable SNP positions among outbreak isolates. Genomic network analyses suggested a single origin of the outbreak, with subsequent division into 3 subclusters. Isolates from patients with confirmed epidemiological links differed by 0-11 SNPs.

Conclusions: Strain-specific SNP genotyping allowed rapid and inexpensive identification of M. tuberculosis outbreak isolates in a population-based strain collection. Subsequent targeted WGS provided detailed insights into transmission dynamics. This combined approach could be applied to track bacterial pathogens in real time and at high resolution.
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http://dx.doi.org/10.1093/infdis/jiu601DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4447836PMC
April 2015

KvarQ: targeted and direct variant calling from fastq reads of bacterial genomes.

BMC Genomics 2014 Oct 9;15:881. Epub 2014 Oct 9.

Swiss Tropical and Public Health Institute, Socinstrasse 57, Basel 4051, Switzerland.

Background: High-throughput DNA sequencing produces vast amounts of data, with millions of short reads that usually have to be mapped to a reference genome or newly assembled. Both reference-based mapping and de novo assembly are computationally intensive, generating large intermediary data files, and thus require bioinformatics skills that are often lacking in the laboratories producing the data. Moreover, many research and practical applications in microbiology require only a small fraction of the whole genome data.

Results: We developed KvarQ, a new tool that directly scans fastq files of bacterial genome sequences for known variants, such as single nucleotide polymorphisms (SNP), bypassing the need of mapping all sequencing reads to a reference genome and de novo assembly. Instead, KvarQ loads "testsuites" that define specific SNPs or short regions of interest in a reference genome, and directly synthesizes the relevant results based on the occurrence of these markers in the fastq files. KvarQ has a versatile command line interface and a graphical user interface. KvarQ currently ships with two "testsuites" for Mycobacterium tuberculosis, but new "testsuites" for other organisms can easily be created and distributed. In this article, we demonstrate how KvarQ can be used to successfully detect all main drug resistance mutations and phylogenetic markers in 880 bacterial whole genome sequences. The average scanning time per genome sequence was two minutes. The variant calls of a subset of these genomes were validated with a standard bioinformatics pipeline and revealed >99% congruency.

Conclusion: KvarQ is a user-friendly tool that directly extracts relevant information from fastq files. This enables researchers and laboratory technicians with limited bioinformatics expertise to scan and analyze raw sequencing data in a matter of minutes. KvarQ is open-source, and pre-compiled packages with a graphical user interface are available at http://www.swisstph.ch/kvarq.
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http://dx.doi.org/10.1186/1471-2164-15-881DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4197298PMC
October 2014

Lipidomics and genomics of Mycobacterium tuberculosis reveal lineage-specific trends in mycolic acid biosynthesis.

Microbiologyopen 2014 Dec 19;3(6):823-35. Epub 2014 Sep 19.

Mycobacterial Division Research, NIMR, MRC, NW71AA, London, United Kingdom; Department of Medical Parasitology and Infection Biology, Swiss TPH, 4002, Basel, Switzerland; University of Basel, 4002, Basel, Switzerland.

Mycolic acids (MAs) are α-alkyl, β-hydroxy long-chain fatty acids found in abundance in the cell envelope of the Mycobacterium tuberculosis complex (MTBC). MAs form an efficient permeability barrier, modulate host innate immune responses, and are the targets of several anti-tuberculosis drugs. Using mass spectrometry, we measured the relative abundance of 80 MA species across 36 clinical isolates of MTBC covering four major phylogenetic lineages. We found significant variations in the MA patterns between different MTBC strains and lineages. MA patterns of "ancient" lineages contrasted those from "modern" lineages, with a lower representation of alpha-mycolates among Lineage 6 strains and an inversion of the methoxy: keto-mycolates ratio in Lineage 1 strains. By interrogating the whole genome sequences of these MTBC strains, we identified relevant single-nucleotide polymorphisms that may sustain the lineage-specific MA patterns. Our results show that the strain genetic background influences MA metabolism and suggests that strain diversity should be considered in the development of new anti-tuberculosis drugs that target MA synthesis.
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http://dx.doi.org/10.1002/mbo3.193DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4263507PMC
December 2014

Impact of in vitro evolution on antigenic diversity of Mycobacterium bovis bacillus Calmette-Guerin (BCG).

Vaccine 2014 Oct 6;32(45):5998-6004. Epub 2014 Sep 6.

Department of Medicine, Division of Infectious Diseases, New York University School of Medicine, 522 First Avenue, Smilow 901, New York, New York, 10016, USA.

Mycobacterium bovis bacillus Calmette-Guerin (BCG), the only vaccine currently used against tuberculosis, is an attenuated derivative of M. bovis that has been propagated in vitro for more than 40 years. We have previously reported that the experimentally-verified human T cell epitopes of the M. tuberculosis complex (MTBC) are the most conserved elements of the genome; whether immune recognition is the force driving the conservation of epitopes in the MTBC is unknown. Therefore, we sequenced the genomes of 12 BCG strains to determine whether T cell epitopes were under selection pressure during BCG in vitro evolution. We constructed a genome-wide phylogeny and refined the previously-determined BCG phylogeny. Notably, we identified a new cluster between BCG Japan and BCG Russia, and repositioned the relationships of several strains within the lineage. We also compared the sequence diversity of 1530 experimentally verified human T cell epitopes in the BCG vaccines with those in the MTBC. We found 23% of the known T cell epitopes are absent, and that the majority (82%) of the absent epitopes in BCG are contained in 6 proteins encoded in 2 regions of difference (RD) unique to BCG strains. We also found that T cell epitope sequences in BCG are more conserved than non-epitope sequences in the same gene. Finally, we find evidence that epitope sequence variation in BCG potentially affects human T cell recognition. These findings provide new insight into sequence variation in a slow-growing bacterium closely related to the MTBC that has been subjected to prolonged passage outside of a mammalian host, and indicate little difference in the extent of variation in vivo and in vitro.
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http://dx.doi.org/10.1016/j.vaccine.2014.07.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4539939PMC
October 2014

Pre-Columbian mycobacterial genomes reveal seals as a source of New World human tuberculosis.

Nature 2014 Oct 20;514(7523):494-7. Epub 2014 Aug 20.

1] Department of Archaeological Sciences, University of Tübingen, Ruemelinstraße 23, 72070 Tübingen, Germany [2] Senckenberg Centre for Human Evolution and Palaeoenvironment, University of Tübingen, Tübingen 72070, Germany [3] Max Planck Institute for Science and History, Khalaische Straße 10, 07745 Jena, Germany.

Modern strains of Mycobacterium tuberculosis from the Americas are closely related to those from Europe, supporting the assumption that human tuberculosis was introduced post-contact. This notion, however, is incompatible with archaeological evidence of pre-contact tuberculosis in the New World. Comparative genomics of modern isolates suggests that M. tuberculosis attained its worldwide distribution following human dispersals out of Africa during the Pleistocene epoch, 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.
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http://dx.doi.org/10.1038/nature13591DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4550673PMC
October 2014

Sequence diversity in the pe_pgrs genes of Mycobacterium tuberculosis is independent of human T cell recognition.

mBio 2014 Jan 14;5(1):e00960-13. Epub 2014 Jan 14.

Unlabelled: The Mycobacterium tuberculosis genome includes the large family of pe_pgrs genes, whose functions are unknown. Because of precedents in other pathogens in which gene families showing high sequence variation are involved in antigenic variation, a similar role has been proposed for the pe_pgrs genes. However, the impact of immune selection on pe_pgrs genes has not been examined. Here, we sequenced 27 pe_pgrs genes in 94 clinical strains from five phylogenetic lineages of the M. tuberculosis complex (MTBC). We found that pe_pgrs genes were overall more diverse than the remainder of the MTBC genome, but individual members of the family varied widely in their nucleotide diversity and insertion/deletion (indel) content: some were more, and others were much less, diverse than the genome average. Individual pe_pgrs genes also differed in the ratio of nonsynonymous to synonymous mutations, suggesting that different selection pressures act on individual pe_pgrs genes. Using bioinformatic methods, we tested whether sequence diversity in pe_pgrs genes might be selected by human T cell recognition, the major mechanism of adaptive immunity to MTBC. We found that the large majority of predicted human T cell epitopes were confined to the conserved PE domain and experimentally confirmed the antigenicity of this domain in tuberculosis patients. In contrast, despite being genetically diverse, the PGRS domains harbored few predicted T cell epitopes. These results indicate that human T cell recognition is not a significant force driving sequence diversity in pe_pgrs genes, which is consistent with the previously reported conservation of human T cell epitopes in the MTBC.

Importance: Recognition of Mycobacterium tuberculosis antigens by T lymphocytes is known to be important for immune protection against tuberculosis, but it is unclear whether human T cell recognition drives antigenic variation in M. tuberculosis. We previously discovered that the known human T cell epitopes in the M. tuberculosis complex are highly conserved, but we hypothesized that undiscovered epitopes with naturally occurring sequence variants might exist. To test this hypothesis, we examined the pe_pgrs genes, a large family of genes that has been proposed to function in immune evasion by M. tuberculosis. We found that the pe_pgrs genes exhibit considerable sequence variation, but the regions containing T cell epitopes and the regions of variation are distinct. These findings confirm that the majority of human T cell epitopes of M. tuberculosis are highly conserved and indicate that selection forces other than T cell recognition drive sequence variation in the pe_pgrs genes.
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http://dx.doi.org/10.1128/mBio.00960-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3903279PMC
January 2014

Mixed infection by Legionella pneumophila in outbreak patients.

Int J Med Microbiol 2014 May 14;304(3-4):307-13. Epub 2013 Nov 14.

Unidad Mixta Genómica y Salud, FISABIO-Salud Pública, Universidad de Valencia, Instituto Cavanilles de Biodiversidad y Biología Evolutiva, Valencia, Spain; CIBER en Epidemiología y Salud Pública (CIBERESP), Spain. Electronic address:

During the molecular epidemiological study of a legionellosis outbreak, we obtained sequence based typing (SBT) profiles from uncultured respiratory samples of 15 affected patients. We detected several distinct allelic profiles some of which were a mixture of alleles present in the more common profiles. Chromatograms from the sequences of one patient with mixed profile showed polymorphisms in several positions, which could result from the simultaneous presence of different Legionella variants in the sample. In order to test this possibility, we cloned PCR amplification products from six loci for two patients with a mixed profile and a patient with a pure profile. After obtaining around 20 sequences for each locus of three patients, we detected several variants in two of them and two variants in the third one. In summary, the three analyzed patients showed evidence of more than one Legionella variant during the acute infection. These results indicate that probably some patients were infected by more than one strain, which could be due to co-infection from the same environmental source or, alternatively, to independent infections in a very short period of time. Although our data cannot discriminate between these hypotheses, these results suggest that Legionella infection patterns can be more complex than previously assumed. None of the environmental samples analyzed during this outbreak was even similar to any of the clinical ones.
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http://dx.doi.org/10.1016/j.ijmm.2013.11.002DOI Listing
May 2014