Improved DNA Amplification of the Hallmark IS900 Element in Mycobacterium avium subsp. paratuberculosis: a Reexamination Based on Whole-Genome Sequence Analysis.

Amplification of the IS900 multicopy element is a hallmark nucleic acid-based diagnostic test for Mycobacterium avium subsp. paratuberculosis, which causes Johne's disease in ruminants. This assay is frequently used to determine the presence of the bacterium in feces of infected cattle and sheep. Two IS900 primer sets developed in the 1990s were widely used for decades, and their use has continued in current studies. However, these primers were developed prior to the availability of complete genome sequences. Recent sequence analysis of the binding locations for one primer pair (P90/P91) identified errors and binding inefficiencies that can be easily corrected to further increase detection sensitivity. The P90 primer is missing two nucleotides that should be present near the 3' end, and it does not bind all copies of IS900 due to 5' deletions at some IS900 loci. These IS900 primer pairs, along with newly developed primers, were tested by real-time PCR on purified genomic DNA to determine which primer set performed the best and how primer design errors affect amplification efficiencies. The newly designed PCR primer set (JB5) showed increased sensitivity by two to three quantification cycles using purified genomic DNA and was similar in efficiency to 150C/921. These tests were extended using DNA from feces and tissues of infected cows, which showed similar results. Finally, a 167-bp partial duplication of IS900 was found in type I strains. Although P90 and P91 primers successfully amplify M. avium subsp. paratuberculosis DNA, their use should be discontinued in favor of more efficient primer pairs in future studies. IMPORTANCE This study is an example of how applied genomic analysis can aid diagnostic test improvements. Detection of Mycobacterium avium subsp. paratuberculosis infection of livestock prior to the appearance of clinical disease signs is very difficult but essential for identifying animals shedding the bacterium to prevent transmission of Johne's disease. Total M. avium subsp. paratuberculosis quantity in the feces as determined by real-time PCR (qPCR) using the IS900 target indicates bacterial shedding status and potential for transmission of the pathogen. However, legacy primers designed prior to the availability of complete genome sequences that are used in these tests to detect M. avium subsp. paratuberculosis were based on data from only a single copy of IS900 and not considering all copies collectively as a group. This approach resulted in primer design errors which can be easily corrected to improve test sensitivities. We tested original primers that contain these errors and their corrected versions by qPCR and showed improved sensitivity on purified genomic DNA as well as fecal and tissue samples. These findings may help detect the organism from environmental samples on farms where sensitivity is currently lacking.

Longitudinal study of Mycobacterium avium ssp. paratuberculosis fecal shedding patterns and concurrent serological patterns in naturally infected dairy cattle.

Mycobacterium avium ssp. paratuberculosis (MAP) is the etiological agent of paratuberculosis, a disease that affects ruminants worldwide. Despite global interest in the control of this disease, gaps exist in our knowledge of fecal shedding patterns and concurrent serological patterns. This longitudinal study in dairy cattle herds with high MAP seroprevalence in France aimed at accurately describing fecal shedding patterns over 1 year; relating those shedding patterns to individual animal characteristics (age, breed, parity); and exploring the association between fecal shedding patterns and serological patterns. To describe temporal fecal shedding patterns and continuity of shedding, along with the standard quantitative PCR (qPCR) threshold cycle we used a cutoff value that related to low or nonculturable fecal shedding. We also defined a threshold cycle indicative of shedding in high quantities to describe infection progression patterns. Twenty-one herds completed the study, and 782 cows were tested 4 times each. We obtained 4 sets of paired fecal qPCR and serum ELISA results from 757 cows. Although we targeted highly likely infectious animals, we found a large diversity of shedding patterns, as well as high variability between herds in the proportion of animals showing a given pattern. The fecal qPCR results of almost 20% of the final study sample were positioned at least once in the range that indicated low or nonculturable fecal shedding (between the adjusted and the standard cutoff value). Although these animals would typically be classified as non-shedders, they could be important to infection dynamics on the farm. Animals that shed at least twice consecutively and animals that shed in high quantities rarely reverted to negativity. Repeated fecal qPCR can be used to detect temporal fecal shedding traits, and the decision to cull an animal could practically be based on temporal, semiquantitative results. Overall, we found a mismatch between fecal shedding and ELISA seropositivity (637 animals were ELISA-negative 4 times, but only 13% of those animals were qPCR-negative 4 times). We found that having more than 2 ELISA-positive samples was strongly related to persistent and continuous shedding. We suggest that although serological testing is much less sensitive than qPCR, it can also be used, particularly over the course of multiple testing events, to identify animals that are most likely to contribute to the contamination of the farm environment.

Cell wall peptidolipids of Mycobacterium avium: from genetic prediction to exact structure of a nonribosomal peptide.

Mycobacteria have a complex cell wall structure that includes many lipids; however, even within a single subspecies of Mycobacterium avium these lipids can differ. Total lipids from an M. avium subsp. paratuberculosis (Map) ovine strain (S-type) contained no identifiable glycopeptidolipids or lipopentapeptide (L5P), yet both lipids are present in other M. avium subspecies. We determined the genetic and phenotypic basis for this difference using sequence analysis as well as biochemical and physico-chemical approaches. This strategy showed that a nonribosomal peptide synthase, encoded by mps1, contains three amino acid specifying modules in ovine strains, compared to five modules in bovine strains (C-type). Sequence analysis predicted these modules would produce the tripeptide Phe-N-Methyl-Val-Ala with a lipid moiety, termed lipotripeptide (L3P). Comprehensive physico-chemical analysis of Map S397 extracts confirmed the structural formula of the native L3P as D-Phe-N-Methyl-L-Val-L-Ala-OMe attached in N-ter to a 20-carbon fatty acid chain. These data demonstrate that S-type strains, which are more adapted in sheep, produce a unique lipid. There is a dose-dependent effect observed for L3P on upregulation of CD25+ CD8 T cells from infected cows, while L5P effects were static. In contrast, L5P demonstrated a significantly stronger induction of CD25+ B cells from infected animals compared to L3P.

Phylogenomic exploration of the relationships between strains of Mycobacterium avium subspecies paratuberculosis.

BACKGROUND: Mycobacterium avium subspecies paratuberculosis (Map) is an infectious enteric pathogen that causes Johne's disease in livestock. Determining genetic diversity is prerequisite to understanding the epidemiology and biology of Map. We performed the first whole genome sequencing (WGS) of 141 global Map isolates that encompass the main molecular strain types currently reported. We investigated the phylogeny of the Map strains, the diversity of the genome and the limitations of commonly used genotyping methods. RESULTS: Single nucleotide polymorphism (SNP) and phylogenetic analyses confirmed two major lineages concordant with the former Type S and Type C designations. The Type I and Type III strain groups are subtypes of Type S, and Type B strains are a subtype of Type C and not restricted to Bison species. We found that the genome-wide SNPs detected provided greater resolution between isolates than currently employed genotyping methods. Furthermore, the SNP used for IS1311 typing is not informative, as it is likely to have occurred after Type S and C strains diverged and does not assign all strains to the correct lineage. Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat (MIRU-VNTR) differentiates Type S from Type C but provides limited resolution between isolates within these lineages and the polymorphisms detected do not necessarily accurately reflect the phylogenetic relationships between strains. WGS of passaged strains and coalescent analysis of the collection revealed a very high level of genetic stability, with the substitution rate estimated to be less than 0.5 SNPs per genome per year. CONCLUSIONS: This study clarifies the phylogenetic relationships between the previously described Map strain groups, and highlights the limitations of current genotyping techniques. Map isolates exhibit restricted genetic diversity and a substitution rate consistent with a monomorphic pathogen. WGS provides the ultimate level of resolution for differentiation between strains. However, WGS alone will not be sufficient for tracing and tracking Map infections, yet importantly it can provide a phylogenetic context for affirming epidemiological connections.

Complete genome sequence of an ovine ancestral strain of Mycobacterium avium subspecies paratuberculosis 6756.

The complete genome sequence of the most ancestral type SI strain of Mycobacterium avium subspecies paratuberculosis 6756, isolated from a sheep, was determined. The genome was sequenced using PacBio technology, yielding a genome size of 4,830,294 nucleotides with no identified plasmids.

Genetic and chemical control of tuberculostearic acid production in Mycobacterium avium subspecies paratuberculosis.

Tuberculostearic acid (TBSA) is a fatty acid unique to mycobacteria and some corynebacteria and has been studied due to its diagnostic value, biofuel properties, and role in membrane dynamics. In this study, we demonstrate that TBSA production can be abrogated either by addition of pivalic acid to mycobacterial growth cultures or by a bfaA gene knockout encoding a flavin adenine dinucleotide (FAD)-binding oxidoreductase. Mycobacterium avium subspecies paratuberculosis (Map) growth and TBSA production were inhibited in 0.5-mg/mL pivalic acid-supplemented cultures, but higher concentrations were needed to have a similar effect in other mycobacteria, including Mycobacterium smegmatis. While Map C-type strains, isolated from cattle and other ruminants, will produce TBSA in the absence of pivalic acid, the S-type Map strains, typically isolated from sheep, do not produce TBSA in any condition. A SAM-dependent methyltransferase encoded by bfaB and FAD-binding oxidoreductase are both required in the two-step biosynthesis of TBSA. However, S-type strains contain a single-nucleotide polymorphism in the bfaA gene, rendering the oxidoreductase enzyme vestigial. This results in the production of an intermediate, termed 10-methylene stearate, which is detected only in S-type strains. Fatty acid methyl ester analysis of a C-type Map bfaA knockout revealed the loss of TBSA production, but the intermediate was present, similar to the S-type strains. Collectively, these results demonstrate the subtle biochemical differences between two primary genetic lineages of Map and other mycobacteria as well as explain the resulting phenotype at the genetic level. These data also suggest that TBSA should not be used as a diagnostic marker for Map.IMPORTANCEBranched-chain fatty acids are a predominant cell wall component among species belonging to the Mycobacterium genus. One of these is TBSA, which is a long-chain middle-branched fatty acid used as a diagnostic marker for Mycobacterium tuberculosis. This fatty acid is also an excellent biolubricant. Control of its production is important for industrial purposes as well as understanding the biology of mycobacteria. In this study, we discovered that a carboxylic acid compound termed pivalic acid inhibits TBSA production in mycobacteria. Furthermore, Map strains from two separate genetic lineages (C-type and S-type) showed differential production of TBSA. Cattle-type strains of Mycobacterium avium subspecies paratuberculosis produce TBSA, while the sheep-type strains do not. This important phenotypic difference is attributed to a single-nucleotide deletion in sheep-type strains of Map. This work sheds further light on the mechanism used by mycobacteria to produce tuberculostearic acid.

Novel feature of Mycobacterium avium subsp. paratuberculosis, highlighted by characterization of the heparin-binding hemagglutinin adhesin.

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.

Interaction of Mycobacterium leprae with human airway epithelial cells: adherence, entry, survival, and identification of potential adhesins by surface proteome analysis.

This study examined the in vitro interaction between Mycobacterium leprae, the causative agent of leprosy, and human alveolar and nasal epithelial cells, demonstrating that M. leprae can enter both cell types and that both are capable of sustaining bacterial survival. Moreover, delivery of M. leprae to the nasal septum of mice resulted in macrophage and epithelial cell infection in the lung tissue, sustaining the idea that the airways constitute an important M. leprae entry route into the human body. Since critical aspects in understanding the mechanisms of infection are the identification and characterization of the adhesins involved in pathogen-host cell interaction, the nude mouse-derived M. leprae cell surface-exposed proteome was studied to uncover potentially relevant adhesin candidates. A total of 279 cell surface-exposed proteins were identified based on selective biotinylation, streptavidin-affinity purification, and shotgun mass spectrometry; 11 of those proteins have been previously described as potential adhesins. In vitro assays with the recombinant forms of the histone-like protein (Hlp) and the heparin-binding hemagglutinin (HBHA), considered to be major mycobacterial adhesins, confirmed their capacity to promote bacterial attachment to epithelial cells. Taking our data together, they suggest that the airway epithelium may act as a reservoir and/or portal of entry for M. leprae in humans. Moreover, our report sheds light on the potentially critical adhesins involved in M. leprae-epithelial cell interaction that may be useful in designing more effective tools for leprosy control.

Genomic epidemiology of Mycobacterium avium subsp. paratuberculosis isolates from Canadian dairy herds provides evidence for multiple infection events.

Mycobacterium avium subsp. paratuberculosis (MAP) is the pathogen responsible for paratuberculosis or Johne's Disease (JD) in ruminants, which is responsible for substantial economic losses worldwide. MAP transmission primarily occurs through the fecal-oral route, and the introduction of an MAP infected animal into a herd is an important transmission route. In the current study, we characterized MAP isolates from 67 cows identified in 20 herds from the provinces of Quebec and Ontario, Canada. Whole genome sequencing (WGS) was performed and an average genome coverage (relative to K-10) of ∼14.9 fold was achieved. The total number of SNPs present in each isolate varied from 51 to 132 and differed significantly between herds. Isolates with the highest genetic variability were generally present in herds from Quebec. The isolates were broadly separated into two main clades and this distinction was not influenced by the province from which they originated. Analysis of 8 MIRU-VNTR loci and 11 SSR loci was performed on the 67 isolates from the 20 dairy herds and publicly available references, notably major genetic lineages and six isolates from the province of Newfoundland and Labrador. All 67 field isolates were phylogenetically classified as Type II (C-type) and according to MIRU-VNTR, the predominant type was INMV 2 (76.1%) among four distinct patterns. Multilocus SSR typing identified 49 distinct INMV SSR patterns. The discriminatory index of the multilocus SSR typing was 0.9846, which was much higher than MIRU-VNTR typing (0.3740). Although multilocus SSR analysis provides good discriminatory power, the resolution was not informative enough to determine inter-herd transmission. In select cases, SNP-based analysis was the only approach able to document disease transmission between herds, further validated by animal movement data. The presence of SNPs in several virulence genes, notably for PE, PPE, mce and mmpL, is expected to explain differential antigenic or pathogenetic host responses. SNP-based studies will provide insight into how MAP genetic variation may impact host-pathogen interactions. Our study highlights the informative power of WGS which is now recommended for epidemiological studies and to document mixed genotypes infections.

Presence of Non-Tuberculous Mycobacteria Including Mycobacterium avium subsp. paratuberculosis Associated with Environmental Amoebae.

One of the obstacles to eradicating paratuberculosis or Johne's Disease (JD) seems to be the persistence of Mycobacterium avium subsp. paratuberculosis (Map) in the environment due to its ability to survive alone or vectorized. It has been shown that Map is widely distributed in soils and water. Previously, we isolated amoebae associated with Map strains in the environment of bovines from an infected herd. This work aims to verify our working hypothesis, which suggests that amoebae may play a role in the transmission of JD. In this study, we sampled water in the vicinity of herds infected with Map or Mycobacterium bovis (M. bovis) and searched for amoebae and mycobacteria. Live amoebae were recovered from all samples. Among these amoebae, four isolates associated with the presence of mycobacteria were identified and characterized. Map and other mycobacterial species were detected by qPCR and, in some cases, by culture. This study suggests that amoebae and Map may be found in the same environment and might represent a risk of exposure of animals to pathogenic mycobacteria. These data open up new perspectives on the control measures to be put in place to prevent contamination by Map.

Deciphering the evolution of the temporal and geographic distribution of French Mycobacterium bovis genotypes using a high throughput SNP-targeted amplicon sequencing method.

Mycobacterium bovis, which belongs to the Mycobacterium tuberculosis complex, is a highly clonal pathogen. However, several lineages of M. bovis have been described worldwide and nine different clusters were identified in France. Targeted amplicon sequencing using next-generation sequencing technology of eighty-eight phylogenetically informative single nucleotide polymorphisms (SNPs) were used to infer the phylogenetic relationship of 630 strains of the National Reference Laboratory isolated between 1979 and 2018 from various animal species. This study allowed classifying 618 different genotypic profiles (combination of a spoligotype and 8 loci-MIRU-VNTR profiles) into the nine previously identified clusters. A global analysis of the entire collection of the National Reference Laboratory has made it possible to represent the evolution of clonal complexes and clusters in time and space for better assessing epidemiological changes of bovine tuberculosis in France.

Features of Mycobacterium bovis Complete Genomes Belonging to 5 Different Lineages.

Mammalian tuberculosis (TB) is a zoonotic disease mainly due to Mycobacterium bovis (M. bovis). A current challenge for its eradication is understanding its transmission within multi-host systems. Improvements in long-read sequencing technologies have made it possible to obtain complete bacterial genomes that provide a comprehensive view of species-specific genomic features. In the context of TB, new genomic references based on complete genomes genetically close to field strains are also essential to perform precise field molecular epidemiological studies. A total of 10 M. bovis strains representing each genetic lineage identified in France and in other countries were selected for performing complete assembly of their genomes. Pangenome analysis revealed a "closed" pangenome composed of 3900 core genes and only 96 accessory genes. Whole genomes-based alignment using progressive Mauve showed remarkable conservation of the genomic synteny except that the genomes have a variable number of copies of IS6110. Characteristic genomic traits of each lineage were identified through the discovery of specific indels. Altogether, these results provide new genetic features that improve the description of M. bovis lineages. The availability of new complete representative genomes of M. bovis will be useful to epidemiological studies and better understand the transmission of this clonal-evolving pathogen.

Characterization of Rosculus vilicus sp. nov., a rhizarian amoeba interacting with Mycobacterium avium subsp. paratuberculosis.

Free-living amoebae are described as potential reservoirs for pathogenic bacteria in the environment. It has been hypothesized that this might be the case for Mycobacterium avium subsp. paratuberculosis, the bacterium responsible for paratuberculosis. In a previous work, we isolated an amoeba from a water sample in the environment of infected cattle and showed that this amoeba was associated with Mycobacterium avium subsp. paratuberculosis. While a partial 18S rRNA gene has allowed us to suggest that this amoeba was Rosculus-like, at that time we were not able to sub-cultivate it. In the present study, we succeeded in cultivating this strain at 20-25°C. This amoeba is among the smallest (5-7 µm) described. The sequencing of the whole genome allowed us to extract the full 18S rRNA gene and propose this strain as a new species of the Rosculus genus, i.e., R. vilicus. Of note, the mitochondrial genome is particularly large (184,954 bp). Finally, we showed that this amoeba was able to phagocyte Mycobacterium avium subsp. paratuberculosis and that the bacterium was still observed within amoebae after at least 3 days. In conclusion, we characterized a new environmental amoeba species at the cellular and genome level that was able to interact with Mycobacterium avium subsp. paratuberculosis. As a result, R. vilicus is a potential candidate as environmental reservoir for Mycobacterium avium subsp. paratuberculosis but further experiments are needed to test this hypothesis.

Inter- and intra-subtype genotypic differences that differentiate Mycobacterium avium subspecies paratuberculosis strains.

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 LSP(A)4 and absence of LSP(A)20 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.

Mycobacterial laminin-binding histone-like protein mediates collagen-dependent cytoadherence.

When grown in the presence of exogenous collagen I, Mycobacterium bovis BCG was shown to form clumps. Scanning electron microscopy examination of these clumps revealed the presence of collagen fibres cross-linking the bacilli. Since collagen is a major constituent of the eukaryotic extracellular matrices, we assayed BCG cytoadherence in the presence of exogenous collagen I. Collagen increased the interaction of the bacilli with A549 type II pneumocytes or U937 macrophages, suggesting that BCG is able to recruit collagen to facilitate its attachment to host cells. Using an affinity chromatography approach, we have isolated a BCG collagen-binding protein corresponding to the previously described mycobacterial laminin-binding histone-like protein (LBP/Hlp), a highly conserved protein associated with the mycobacterial cell wall. Moreover, Mycobacterium leprae LBP/Hlp, a well-characterized adhesin, was also able to bind collagen I. Finally, using recombinant fragments of M. leprae LBP/Hlp, we mapped the collagen-binding activity within the C-terminal domain of the adhesin. Since this protein was already shown to be involved in the recognition of laminin and heparan sulphate-containing proteoglycans, the present observations reinforce the adhesive activities of LBP/Hlp, which can be therefore considered as a multifaceted mycobacterial adhesin, playing an important role in both leprosy and tuberculosis pathogenesis.

IS6110 Copy Number in Multi-Host Mycobacterium bovis Strains Circulating in Bovine Tuberculosis Endemic French Regions.

IS6110 is an insertion sequence found in the Mycobacterium tuberculosis complex, to which Mycobacterium bovis belongs, which can play a role in genome plasticity and in bacterial evolution. In this study, the abundance and location of IS6110 on M. bovis genomic data of French animal field strains were studied. A first analysis was performed on a panel of 81 strains that reflect the national M. bovis population's genetic diversity. The results show that more than one-third of them are IS6110 multicopy and that 10% have IS6110 in a high copy number (more than 6 copies). Multicopy strains are those circulating in the regions where prevalence was above the national average. Further study of 93 such strains, with an IS6110 copy number of 10-12, showed stability of IS6110 copy number and genome location over time and between host species. The correlation between M. bovis multicopy strains and high bovine tuberculosis (bTB) prevalence leads us to consider whether their epidemiological success could be partly due to genetic changes originated by IS6110 transposition.

Designing quinoline-isoniazid hybrids as potent anti-tubercular agents inhibiting mycolic acid biosynthesis.

Isoniazid is a cornerstone of modern tuberculosis (TB) therapy and targets the enoyl ACP reductase InhA, a key enzyme in mycolic acid biosynthesis. InhA is still a promising target for the development of new anti-TB drugs. Herein, we report the design, synthesis, and anti-tubercular activity of new isoniazid hybrids. Among these, 1H-1,2,3 triazole-tethered quinoline-isoniazid conjugates 16a to 16g exhibited high activity against Mycobacterium tuberculosis with minimal inhibitory concentrations in the 0.25-0.50 µg/mL range and were bactericidal in vitro. Importantly, these compounds were well tolerated at high doses on mammalian cells, leading to high selectivity indices. The hybrids were dependent on functional KatG production to inhibit mycolic acid biosynthesis. Moreover, overexpression of InhA in M. tuberculosis resulted in high resistance levels to 16a-16g and reduced mycolic acid biosynthesis inhibition, similar to isoniazid. Overall, these findings suggest that the synthesized quinoline-isoniazid hybrids are promising anti-tubercular molecules, which require further pre-clinical evaluation.

Genetic Features of Mycobacterium avium subsp. paratuberculosis Strains Circulating in the West of France Deciphered by Whole-Genome Sequencing.

Paratuberculosis is a chronic infection of the intestine, mainly the ileum, caused by Mycobacterium avium subsp. paratuberculosis in cattle and other ruminants. This enzootic disease is present worldwide and has a negative impact on the dairy cattle industry. For this subspecies, the current genotyping tools do not provide the needed resolution to investigate the genetic diversity of closely related strains. These limitations can be overcome by the application of whole-genome sequencing (WGS), particularly for clonal populations such as M. avium subsp. paratuberculosis. The purpose of the present study was to undertake a WGS analysis with a panel of 200 animal field M. avium subsp. paratuberculosis strains selected based on a previous large-scale longitudinal study of Prim'Holstein and Normande dairy breeds naturally infected with M. avium subsp. paratuberculosis in the West of France. The pangenome analysis revealed that M. avium subsp. paratuberculosis has a closed pangenome. The phylogeny, based on alignment of 2,786 nonhomoplasic single nucleotide polymorphisms (SNPs), showed that the strain population is structured into three clades independently of the cattle breed or geographic distribution. The increased resolution of phylogeny obtained by WGS confirmed the homoplasic nature of the markers variable-number tandem repeat (VNTR) and short sequence repeat (SSR) used for M. avium subsp. paratuberculosis genotyping. These phylogenetic data also revealed independent introductions of the different genotypes in two main waves since at least 2003. WGS applied to this sampling demonstrated the presence of mixed infections in herds and at the individual animal level. Collectively, the phylogeny results inferred with French isolates compared to M. avium subsp. paratuberculosis isolates from around the world suggest introductions of M. avium subsp. paratuberculosis genotypes through the animal trade. Relationships between genetic traits and epidemiological data can now be investigated to better understand transmission dynamics of the disease. IMPORTANCE Mycobacterium avium subsp. paratuberculosis causes Johne's disease in ruminants, which is present worldwide and has significant negative impacts on the dairy cattle industry and animal welfare. Prevention and control of M. avium subsp. paratuberculosis infection are hampered by knowledge gaps in strain virulence, genotype distribution, and transmission dynamics. This work has revealed new insights into M. avium subsp. paratuberculosis strains currently circulating in western France and how they are related to strains circulating globally. We applied whole-genome sequencing (WGS) to obtain comprehensive information on genome evolution and discrimination of closely related strains. This approach revealed the history of M. avium subsp. paratuberculosis infection in France, refined the pangenomic characteristics of M. avium subsp. paratuberculosis, and demonstrated the existence of mixed infection in animals. Finally, this study identified predominant genotypes, which allow a better understanding of disease transmission dynamics. This information will facilitate tracking of this pathogen on farms and across agricultural regions, thus informing transmission pathways and disease control points.

Immunogenicity of Mycobacterium avium subsp. paratuberculosis proteins in Crohn's disease patients.

UNLABELLED: The association between Mycobacterium avium subsp. paratuberculosis (MAP) and Crohn's disease (CD) is supported by several studies reporting the detection or isolation of MAP from human tissues, but a direct association is still debatable. OBJECTIVE: To evaluate the survival of MAP in human intestinal cells and to measure the presence of antibodies against two mycobacterial proteins necessary for the survival of the bacterium in the sera of CD patients. MATERIAL AND METHODS: Human-derived intestinal cells were infected with three isolates of MAP and the survival of the microorganism was determined. The presence of antibodies against protein tyrosine phosphatase A (PtpA) and protein kinase G (two proteins secreted within the host in the early stages of the invasion) in the sera of CD patients was evaluated. Sera of 20 CD patients and 20 controls were collected and the presence of the antibodies was assayed using enzyme-linked immunosorbent assay (ELISA). Secretion of the PtpA in vivo was visualized by immunostaining. RESULTS: MAP survived in intestinal cells, and immunostaining of PtpA showed that the protein was secreted within these cells. Wilcoxon rank sum test revealed that CD patient sera had significantly higher titer of antibodies specific for both of these antigens compared to controls. ELISA results for either protein were not statistically different between men and women. CONCLUSIONS: The presence of specific antibodies against mycobacterial proteins essential for establishing an infection in the host suggests that MAP can potentially be active in CD patients, and a serological test can be developed for early detection of MAP in CD patients.

Serum antibodies to Mycobacterium avium subspecies paratuberculosis combined with anti-Saccharomyces cerevisiae antibodies in Crohn's disease patients: prevalence and diagnostic role.

BACKGROUND: Because Mycobacterium avium subspecies paratuberculosis (MAP), the etiologic agent of Johne's disease in ruminant, has been identified in the mucosal layer and deeper bowel wall in CD patients, the seroactivity against MAP may define a distinct subset of patients requiring individual treatment. The aim of this study was to assess the performance of anti-MAP antibodies in the diagnostic strategy for CD. METHODS: Two hundred seventy-two individuals were included: 81 with CD, 36 with ulcerative colitis, 35 with coeliac diseases and 120 healthy blood donors. Anti-MAP were detected by ELISA using a purified protein derivative from MAP. Anti-Saccharomyces cerevisiae antibodies (ASCA) were detected by indirect immunofluorescence. RESULTS: The sensitivity and specificity of anti-MAP and ASCA for CD diagnosis were similar (sensitivity: 0.33 ± 0.10 and 0.31 ± 0.10; specificity: 0.96 ± 0.03 and 0.98 ± 0.02, respectively). A combination of these two tests enabled an increase in sensitivity (0.53 ± 0.10), although specificity remained unchanged (0.95 ± 0.04). No correlation was found between anti-MAP positivity and clinical features such as age at onset and the duration of CD, disease location, or intestinal complications. Conversely, extra-intestinal manifestations of CD were statistically associated with a positivity of anti-MAP (48% vs. 24%, P = 0.028), mostly with respect to arthritis (44.5% vs. 13%, P < 0.002). Interestingly, anti-MAP and ASCA were also found in an active form of coeliac disease. CONCLUSION: Our results suggest a complementary role of ASCA and anti-MAP for CD diagnosis and a possible common role of bacteria in small intestinal mucosal damage in CD and coeliac disease.