Regulation of Mycobacterium biofilm development and novel measures against antibiotics resistance.

Currently, there are over 170 recognized species of Mycobacterium, the only genus in the family Mycobacteriaceae. Organisms belonging to this genus are quite diverse with respect to their ability to cause disease in humans. The Mycobacterium genus includes human pathogens (Mycobacterium tuberculosis complex and Mycobacterium leprae) and environmental microorganisms known as non-tuberculosis mycobacteria (NTM). A common pathogenic factor of Mycobacterium is the formation of biofilms. Bacterial biofilms are usually defined as bacterial communities attached to the surface, and are also considered as shared spaces of encapsulated microbial cells, including various extracellular polymeric substrates (EPS), such as polysaccharides, proteins, amyloid proteins, lipids, and extracellular DNA (EDNA), as well as membrane vesicles and humic like microorganisms derived refractory substances. The assembly and dynamics of the matrix are mainly coordinated by second messengers, signaling molecules, or small RNAs. Fully deciphering how bacteria provide structure for the matrix, thereby promoting extracellular reactions and benefiting from them, remains a challenge for future biofilm research. This review introduces a five step development model for biofilms and a new model for biofilm formation, analyses the pathogenicity of biofilms, their interactions with bacteriophages and host immune cells, and the key genes and regulatory networks of mycobacterial biofilms, as well as mycobacterial biofilms and drug resistance, in order to provide a basis for clinical treatment of diseases caused by biofilms.

Challenges and advances in serological and molecular tests to aid leprosy diagnosis.

Leprosy is a neglected chronic infectious disease caused by obligate intracellular bacilli, Mycobacterium leprae and Mycobacterium lepromatosis. Despite multidrug therapy (MDT) success, leprosy accounts for more than 200,000 new cases yearly. Leprosy diagnosis remains based on the dermato-neurologic examination, but histopathology of skin biopsy and bacilloscopy of intradermal scraping are subsidiary diagnostic tests that require expertise and laboratory infrastructure. This minireview summarizes the state of the art of serologic tests to aid leprosy diagnosis, highlighting enzyme-linked immunosorbent assay (ELISA) and point-of-care tests (POCT) biotechnologies. Also, the impact of the postgenomic era on the description of new recombinantly expressed M. leprae-specific protein antigens, such as leprosy Infectious Disease Research Institute (IDRI) diagnostic (LID)-1 is summarized. Highly specific and sensitive molecular techniques to detect M. leprae DNA as the quantitative polymerase chain reaction (qPCR) and the loop-mediated isothermal amplification (LAMP) are briefly reviewed. Serology studies using phenolic glycolipid-I (PGL-I) semi-synthetic antigens, LID-1 fusion antigen, and the single fusion complex natural disaccharide-octyl (NDO)-LID show high sensitivity in multibacillary (MB) patients. However, serology is not applicable to paucibacillary patients, as they have weak humoral response and robust cell-mediated response, requiring tests for cellular biomarkers. Unlike ELISA-based tests, leprosy-specific POCT based on semi-synthetic PGL-I antigens and NDO-LID 1 antigen is easy to perform, cheaper, equipment-free, and can contribute to early diagnosis avoiding permanent incapacities and helping to interrupt M. leprae transmission. Besides its use to help diagnosis of household contacts or at-risk populations in endemic areas, potential applications of leprosy serology include monitoring MDT efficacy, identification of recent infection, especially in young children, as surrogate markers of disease progression to orient adult chemoprophylaxis and as a predictor of type 2 leprosy reactions. Advances in molecular biology techniques have reduced the complexity and execution time of qPCR confirming its utility to help diagnosis while leprosy-specific LAMP holds promise as an adjunct test to detect M. leprae DNA.

Highly sensitive and rapid determination of Mycobacterium leprae based on real-time multiple cross displacement amplification.

BACKGROUND: Mycobacterium leprae (ML) is the pathogen that causes leprosy, which has a long history and still exists today. ML is an intracellular mycobacterium that dominantly induces leprosy by causing permanent damage to the skin, nerves, limbs and eyes as well as deformities and disabilities. Moreover, ML grows slowly and is nonculturable in vitro. Given the prevalence of leprosy, a highly sensitive and rapid method for the early diagnosis of leprosy is urgently needed. RESULTS: In this study, we devised a novel tool for the diagnosis of leprosy by combining restriction endonuclease, real-time fluorescence analysis and multiple cross displacement amplification (E-RT-MCDA). To establish the system, primers for the target gene RLEP were designed, and the optimal conditions for E-RT-MCDA at 67 °C for 36 min were determined. Genomic DNA from ML, various pathogens and clinical samples was used to evaluate and optimize the E-RT-MCDA assay. The limit of detection (LoD) was 48.6 fg per vessel for pure ML genomic DNA, and the specificity of detection was as high as 100%. In addition, the detection process could be completed in 36 min by using a real-time monitor. CONCLUSION: The E-RT-MCDA method devised in the current study is a reliable, sensitive and rapid technique for leprosy diagnosis and could be used as a potential tool in clinical settings.

In diverse conditions, intrinsic chromatin condensates have liquid-like material properties.

Nuclear DNA in eukaryotes is wrapped around histone proteins to form nucleosomes on a chromatin fiber. Dynamic folding of the chromatin fiber into loops and variations in the degree of chromatin compaction regulate essential processes such as transcription, recombination, and mitotic chromosome segregation. Our understanding of the physical properties that allow chromatin to be dynamically remodeled even in highly compacted states is limited. Previously, we reported that chromatin has an intrinsic capacity to phase separate and form dynamic liquid-like condensates, which can be regulated by cellular factors [B. A. Gibson et al., Cell 179, 470-484.e421 (2019)]. Recent contradictory reports claim that a specific set of solution conditions is required for fluidity in condensates that would otherwise be solid [J. C. Hansen, K. Maeshima, M. J. Hendzel, Epigenetics Chromatin 14, 50 (2021); H. Strickfaden et al., Cell 183, 1772-1784.e1713 (2020)]. We sought to resolve these discrepancies, as our ability to translate with confidence these biophysical observations to cells requires their precise characterization. Moreover, whether chromatin assemblies are dynamic or static affects how processes such as transcription, loop extrusion, and remodeling will engage them inside cells. Here, we show in diverse conditions and without specific buffering components that chromatin fragments form phase separated fluids in vitro. We also explore how sample preparation and imaging affect the experimental observation of chromatin condensate dynamics. Last, we describe how liquid-like in vitro behaviors can translate to the locally dynamic but globally constrained chromatin movement observed in cells.

Sensitivity of different DNA extraction methods and PCR to detect resistance in patients with leprosy stratified by the bacilloscopic index.

INTRODUCTION: Antimicrobial resistance in leprosy is an emerging problem, and the quantitative impact of low bacilloscopic indexes (BIs) on the sensitivity of molecular tests is unknown. We aimed to evaluate the sensitivity of gene sequencing for the detection of mutations related to antimicrobial resistance in Mycobacterium leprae in patients with low BIs using an analytical model. METHODS: Patients with leprosy were included and divided into two groups depending on their BIs (≥ 2+ and < 2+). The sensitivities of the two DNA extraction methods were compared after amplifying and sequencing the repetitive element (RLEP), folP1, rpoB and gyrA in M. leprae. RESULTS: We included 56 patients with leprosy: 35 had BIs less than 2+ (22 had negative slit-skin smear [SSS] results) and 21 patients had BIs greater than or equal to 2+. The sensitivity of the amplification of the RLEP target and the gene sequencing of folP1, rpoB and gyrA was 50 to 70% lower in patients with a BI less than 2+ and was significantly reduced in patients with lower BIs for all targets (p < 0.001). One patient had a mutation in the folP1 gene, and 14 patients had mutations in the gyrA gene, but no mutations related to antimicrobial resistance were found. CONCLUSIONS: We can conclude that the sensitivity of molecular tests is directly related to the BI, but these tests can still detect up to 20% of the targets in patients with BIs < 2+. New strategies to improve the sensitivity for detecting antimicrobial resistance in leprosy patients and reasonable clinical criteria for follow-up and the introduction of alternative treatments must be developed.

Taxonomy of Diamesa steinboecki group (Diptera: Chironomidae: Diamesinae), with description and DNA barcoding of known species. II. Subgroups davisi, leona and loeffleri.

Illustrated redescription of the adult males Diamesa alpina Tokunaga from the Russian Far East and North America, D. amplexivirilia Hansen from Arctic and the Russian Far East, D. saetheri Willassen from Chukotka Region and Kolyma River basin, D. lupus Willassen from Alaska, D. serratosioi Willassen from Norway and Russian Far East, D. leoniella Hansen from Alaska, D. leona Roback from Eurasia, D. japonica Tokunaga from Japan and Russian Far East, D. khumbugelida Saether et Willassen and D. loeffleri Reiss from Himalayas are provided. Morphological data and DNA sequences of the mitochondrial cytochrome c oxidase subunit I gene (COI) were used to delimit of seven species from subgroups davisi (D. alpina, D. amplexivirilia, D. serratosioi), leona (D. japonica, D. leona) and loeffleri (D. khumbugelida, D. loeffleri). Taxonomic remarks with data on geographical distribution of the investigated species are given.

Diagnostic Utility of Biplex/Multiplex Polymerase Chain Reaction in Infectious Granulomatous Dermatitis in North Indian Population.

BACKGROUND: A definite diagnosis of infectious granulomatous dermatitis (IGD) is difficult for both practicing dermatologists and dermatopathologists due to overlapping clinical and histomorphological features. We aimed to explore the role of multiplex polymerase chain reaction (PCR) for identifying a definite etiological agent for diagnosis and appropriate treatment in IGD in formalin-fixed paraffin-embedded tissue. MATERIALS AND METHODS: Sixty-two cases of IGD were included, excluding leprosy. The histochemical stains including Ziehl-Neelsen, periodic acid-Schiff, and Giemsa were performed in all cases. A multiplex PCR was designed for detection of tuberculosis (TB) (IS6110 and mpt64), fungal infections (ITS1, ITS2; ZM1, and ZM3), and leishmaniasis (kDNA). The results of histomorphology, histochemical stains, and multiplex PCR were compared. RESULTS: Among 62 cases, the sensitivity rate of PCR detection for organisms was 16.7%, 0%, 100%, 72%, 75%, and 66.7% in patients with TB, suggestive of TB, leishmaniasis, fungal infections, and granulomatous dermatitis not otherwise specified and granulomatous dermatitis suggestive of fungus, respectively. The TB PCR using IS6110 primers was negative in all cases; however, PCR using mpt64 primers was positive in 33.33% cases of scrofuloderma. The histochemical stains including Ziehl-Neelsen for acid-fast bacilli, periodic acid-Schiff for fungus, and Giemsa for Leishman-Donovan bodies showed positivity in 11.3%, 43.5%, and 3.2%, respectively. CONCLUSION: A multiplex PCR (Mycobacterium tuberculosis, Leishmania, and panfungal) is highly recommended in all cases of IGD where an etiological agent is difficult to establish by skin biopsy and histochemical stains along with a clinicopathological correlation. This will augment in appropriate treatment and will reduce empirical treatment and morbidity in such patients.

DNA minor groove binding of a well known anti-mycobacterial drug dapsone: A spectroscopic, viscometric and molecular docking study.

Dapsone is a sulfone drug mainly used as anti-microbial and anti-inflammatory agent for the treatment of various diseases including leprosy. Recently, its interaction with protein (bovine serum albumin) is evidenced. But, the binding propensity of this anti-mycobacterial drug towards DNA is still unknown. Also, the mode of dapsone-DNA interaction (if any) is still an unknown quantity. In this study, we have taken a thorough attempt to understand these two unknown aspects using various biophysical and in silico molecular docking techniques. Both UV-visible and fluorescence titrimetric studies indicated that dapsone binds to CT-DNA with a binding constant in order of 104 M-1. Circular dichroism, thermal denaturation and viscosity experiments revealed that dapsone binds to the grooves of CT-DNA. Competitive DNA binding studies clearly indicated the minor groove binding property of this anti-mycobacterial drug. Molecular docking provided detailed information about the formation of hydrogen bonding in the dapsone-DNA complex. This in silico study further revealed that dapsone binds to the AT-rich region of the minor groove of DNA having a relative binding energy of -6.22 kcal mol-1. Overall, all these findings evolved from this study can be used for better understanding the medicinal importance of dapsone.

Depicting the DNA binding and photo-nuclease ability of anti-mycobacterial drug rifampicin: A biophysical and molecular docking perspective.

Rifampicin, an important member of ansamycin family, exhibits various biological activities. It is frequently used for the treatment of tuberculosis and leprosy. Recently, its interaction with protein is evidenced. But, its interaction with DNA is still unknown. Whether, exhibition of anti-cancer activity of rifampicin is associated with DNA-cleavage activity is also unknown. In this study, an attempt has been taken to understand these two unknown aspects. Spectroscopic studies indicated that rifampicin binds to CT-DNA with a binding constant of ~5.22 × 105 M-1. Several independent experiments like CD analysis, competitive displacement experiments and viscosity measurements revealed that rifampicin intercalates into the CT-DNA. Molecular docking studies corroborate this fact and depicted that this drug binds to the GC-rich region of DNA through multiple hydrogen bonding having the relative binding energy of -9.21 kcal mol-1. Besides, DNA binding ability, rifampicin causes the photo-cleavage of pUC19 DNA via singlet oxygen pathway. To the best of our knowledge, we report for the first time the DNA binding and DNA cleavage ability of rifampicin. This study provides a clue behind the execution of the anti-cancer activity of rifampicin. Overall, all these information can be used for further understanding the pharmacological effects of rifampicin.

Characterization of Ofloxacin Interaction with Mutated (A91V) Quinolone Resistance Determining Region of DNA Gyrase in Mycobacterium Leprae through Computational Simulation.

Mycobacterium leprae, the causal agent of leprosy is non-cultivable in vitro. Thus, the assessment of antibiotic activity against Mycobacterium leprae depends primarily upon the time-consuming mouse footpad system. The GyrA protein of Mycobacterium leprae is the target of the antimycobacterial drug, Ofloxacin. In recent times, the GyrA mutation (A91V) has been found to be resistant to Ofloxacin. This phenomenon has necessitated the development of new, long-acting antimycobacterial compounds. The underlying mechanism of drug resistance is not completely known. Currently, experimentally crystallized GyrA-DNA-OFLX models are not available for highlighting the binding and mechanism of Ofloxacin resistance. Hence, we employed computational approaches to characterize the Ofloxacin interaction with both the native and mutant forms of GyrA complexed with DNA. Binding energy measurements obtained from molecular docking studies highlights hydrogen bond-mediated efficient binding of Ofloxacin to Asp47 in the native GyrA-DNA complex in comparison with that of the mutant GyrA-DNA complex. Further, molecular dynamics studies highlighted the stable binding of Ofloxacin with native GyrA-DNA complex than with the mutant GyrA-DNA complex. This mechanism provided a plausible reason for the reported, reduced effect of Ofloxacin to control leprosy in individuals with the A91V mutation. Our report is the first of its kind wherein the basis for the Ofloxacin drug resistance mechanism has been explored with the help of ternary Mycobacterium leprae complex, GyrA-DNA-OFLX. These structural insights will provide useful information for designing new drugs to target the Ofloxacin-resistant DNA gyrase.

Ensuring due process in the IACUC and animal welfare setting: considerations in developing noncompliance policies and procedures for institutional animal care and use committees and institutional officials.

Every institution that is involved in research with animals is expected to have in place policies and procedures for the management of allegations of noncompliance with the Animal Welfare Act and the U.S. Public Health Service Policy on the Humane Care and Use of Laboratory Animals. We present here a model set of recommendations for institutional animal care and use committees and institutional officials to ensure appropriate consideration of allegations of noncompliance with federal Animal Welfare Act regulations that carry a significant risk or specific threat to animal welfare. This guidance has 3 overarching aims: 1) protecting the welfare of research animals; 2) according fair treatment and due process to an individual accused of noncompliance; and 3) ensuring compliance with federal regulations. Through this guidance, the present work seeks to advance the cause of scientific integrity, animal welfare, and the public trust while recognizing and supporting the critical importance of animal research for the betterment of the health of both humans and animals.-Hansen, B. C., Gografe, S., Pritt, S., Jen, K.-L. C., McWhirter, C. A., Barman, S. M., Comuzzie, A., Greene, M., McNulty, J. A., Michele, D. E., Moaddab, N., Nelson, R. J., Norris, K., Uray, K. D., Banks, R., Westlund, K. N., Yates, B. J., Silverman, J., Hansen, K. D., Redman, B. Ensuring due process in the IACUC and animal welfare setting: considerations in developing noncompliance policies and procedures for institutional animal care and use committees and institutional officials.

Double differential cross sections for proton induced electron emission from molecular analogues of DNA constituents for energies in the Bragg peak region.

For track structure simulations in the Bragg peak region, measured electron emission cross sections of DNA constituents are required as input for developing parameterized model functions representing the scattering probabilities. In the present work, double differential cross sections were measured for the electron emission from vapor-phase pyrimidine, tetrahydrofuran, and trimethyl phosphate that are structural analogues to the base, the sugar, and the phosphate residue of the DNA, respectively. The range of proton energies was from 75 keV to 135 keV, the angles ranged from 15° to 135°, and the electron energies were measured from 10 eV to 200 eV. Single differential and total electron emission cross sections are derived by integration over angle and electron energy and compared to the semi-empirical Hansen-Kocbach-Stolterfoht (HKS) model and a quantum mechanical calculation employing the first Born approximation with corrected boundary conditions (CB1). The CB1 provides the best prediction of double and single differential cross section, while total cross sections can be fitted with semi-empirical models. The cross sections of the three samples are proportional to their total number of valence electrons.

DNA Sensing via TLR-9 Constitutes a Major Innate Immunity Pathway Activated during Erythema Nodosum Leprosum.

The chronic course of lepromatous leprosy may be interrupted by acute inflammatory episodes known as erythema nodosum leprosum (ENL). Despite its being a major cause of peripheral nerve damage in leprosy patients, the immunopathogenesis of ENL remains ill-defined. Recognized by distinct families of germline-encoded pattern recognition receptors, endogenous and pathogen-derived nucleic acids are highly immunostimulatory molecules that play a major role in the host defense against infections, autoimmunity, and autoinflammation. The aim of this work was to investigate whether DNA sensing via TLR-9 constitutes a major inflammatory pathway during ENL. Flow cytometry and immunohistochemistry analysis showed significantly higher TLR-9 expression in ENL when compared with nonreactional lepromatous patients, both locally in the skin lesions and in circulating mononuclear cells. The levels of endogenous and pathogen-derived TLR-9 ligands in the circulation of ENL patients were also higher. Furthermore, PBMCs isolated from the ENL patients secreted higher levels of TNF, IL-6, and IL-1ß in response to a TLR-9 agonist than those of the nonreactional patients and healthy individuals. Finally, E6446, a TLR-9 synthetic antagonist, was able to significantly inhibit the secretion of proinflammatory cytokines by ENL PBMCs in response to Mycobacterium leprae lysate. Our data strongly indicate that DNA sensing via TLR-9 constitutes a major innate immunity pathway involved in the pathogenesis and evolution of ENL. Thus, the use of TLR-9 antagonists emerges as a potential alternative to more effectively treat ENL aiming to prevent the development of nerve injuries and deformities in leprosy.

Diagnóstico diferencial de tuberculosis en base a PCR en lesiones granulomatosas clasificadas histopatológicamente / PCR-based differential diagnosis of tuberculosis in histopathologically classified granulomatous lesions

El diagnóstico diferencial de tuberculosis en tejidos fijados en formalina e incluidos en parafina es necesario porque la morfología de la lesión tuberculosa es variada, hay diversos granulomas clasificados en necrobióticos, tuberculoideos, supurativos, sarcoideo, a cuerpo extraño/crónico inespecífico. Las lesiones granulomatosas ocurren en tuberculosis y también en otras infecciones (hongos, parásitos, brucelosis, lepra) en condiciones tóxicas, alérgicas, autoinmunes, tumores y otras. El diagnóstico histológico no es confirmatorio de tuberculosis y en ausencia de una baciloscopia positiva, se hace necesaria la confirmación molecular para el diagnóstico diferencial. Evaluamos la eficacia de la técnica de PCR para la detección de tuberculosis en tejidos fijados y comparamos esos resultados con la histología del granuloma y la baciloscopia. Analizamos 444 biopsias de diferentes tejidos (ganglios, piel, pleura, pulmón, intestino, tejido óseo, mama y otros) de 5 tipos de granulomas: G1.tuberculoideo con necrosis caseosa; G2.tuberculoideo sin necrosis caseosa; G3. supurativo; G4. sarcoideo l; G5. a cuerpo extraño/inespecífico. Utilizamos dos PCR-IS6110 nested para detección del complejo Mycobacterium tuberculosis y un pan PCR-hsp65 nested para detección de Mycobacterium spp. Los resultados obtenidos muestran que la detección de tuberculosis mediante PCR fue significativamente superior que mediante baciloscopia. G1: PCR 69,6%, baciloscopia 31,3%; G2: PCR 26,8%, baciloscopia 6,1%; G3: PCR 16,7%, baciloscopia 6,7%; G4: PCR 7%, baciloscopia 4%; G5: PCR 6,7%, baciloscopia 0%. Concluimos que el diagnóstico molecular de tuberculosis mediante un PCR robusto adaptado a tejidos fijados es eficaz, rápido, sensible y contribuye a la precisión del diagnóstico diferencial en diferentes tipos de granulomas (AU)

The differential diagnosis of tuberculosis in fixed paraffin embedded-tissues is necessary due to both the diverse morphology of tuberculous lesions and the varying histological types of granulomas (necrobiotic, tuberculoid, suppurative, sarcoidal and foreign body/inespecific). Granulomatous lesions occur in tuberculosis, in other infections (fungal, parasitic, brucelosis, lepra), in toxic, allergic and autoimmune, tumours and in conditions of unknown etiology. Diagnosis of tuberculosis cannot be confirmed by histopathology alone and in absence of a positive acid-fast bacilli (AFB) stain, molecular confirmation of tuberculosis is necessary for a correct differential diagnosis. The aim of our study was to assess PCR efficacy for mycobacterial infection detection in fixed tissues and to correlate those findings with granuloma histology and with AFB staining. We analyzed 444 biopsies from various tissues (lymph nodes, skin, pleura, lung, intestine, bone tissue, breast and others) with 5 granuloma types: G1: with caseous necrosis; G2: without caseous necrosis; G3: suppurative; G4: sarcoidal; G5: chronic/nonspecific. For molecular detection, we used nested PCR-IS6110 for Mycobacterium tuberculosis complex and a nested pan PCR-hsp65 for Mycobacterium sp.. The results obtained demonstrated that PCR was significantly better than AFB stain for tuberculosis detection. G1: PCR 69.6%, AFB staining 31.3%. G2: PCR 26.8%, AFB staining 6.1%; G3: PCR 16.7%, AFB staining 6.7%; G4: PCR 7%, AFB staining 4%. G5: PCR 6.7%, AFB staining 0%. We conclude that molecular diagnosis of tuberculosis using robust PCR-based testing adapted to fixed tissues is a fast, efficient and sensitive method that increases the accuracy of the differential diagnosis of granulomatous lesions (AU)

Palaeopathology and genes: investigating the genetics of infectious diseases in excavated human skeletal remains and mummies from past populations.

The aim of this paper is to review the use of genetics in palaeomicrobiology, and to highlight the importance of understanding past diseases. Palaeomicrobiology is the study of disease pathogens in skeletal and mummified remains from archaeological contexts. It has revolutionarised our understanding of health in the past by enabling a deeper knowledge of the origins and evolution of many diseases that have shaped us as a species. Bacterial diseases explored include tuberculosis, leprosy, bubonic plague, typhoid, syphilis, endemic and epidemic typhus, trench fever, and Helicobacter pylori. Viral diseases discussed include influenza, hepatitis B, human papilloma virus (HPV), human T-cell lymphotrophic virus (HTLV-1) and human immunodeficiency virus (HIV). Parasitic diseases investigated include malaria, leishmaniasis, Chagas' disease, roundworm, whipworm, pinworm, Chinese liver fluke, fleas and lice. Through a better understanding of disease origins and their evolution, we can place into context how many infectious diseases are changing over time, and so help us estimate how they may change in the future.