Dielectric properties of human macrophages are altered by Mycobacterium tuberculosis infection.

The analysis of cell electrophysiology for pathogenic samples at BSL3 can be problematic. It is virtually impossible to isolate infected from uninfected without a label, for example green fluorescent protein, which can potentially alter the cell electrical properties. Furthermore, the measurement of highly pathogenic organisms often requires equipment dedicated only for use with these organisms due to safety considerations. To address this, we have used dielectrophoresis to study the electrical properties of the human THP-1 cell line and monocyte-derived macrophages before and after infection with non-labelled Mycobacterium tuberculosis. Infection with these highly pathogenic bacilli resulted in changes including a raised surface conductance (associated with reduced zeta potential) and increased capacitance, suggesting an increase in surface roughness. We have also investigated the effect of fixation on THP-1 cells as a means to enable study on fixed samples in BSL1 or 2 laboratories, which suggests that the properties of these cells are largely unaffected by the fixation process. This advance results in a novel technique enabling the isolation of infected and non-infected cells in a sample without labelling.

Oxygen evolution from extremophilic cyanobacteria confined in hard biocoatings.

Biocoatings, in which viable bacteria are immobilized within a waterborne polymer coating for a wide range of potential applications, have garnered greater interest in recent years. In bioreactors, biocoatings can be ready-to-use alternatives for carbon capture or biofuel production that could be reused multiple times. Here, we have immobilized cyanobacteria in mechanically hard biocoatings, which were deposited from polymer colloids in water (i.e., latex). The biocoatings are formed upon heating to 37°C and fully dried before rehydrating. The viability and oxygen evolution of three cyanobacterial species within the biocoatings were compared. Synechococcus sp. PCC 7002 was non-viable inside the biocoatings immediately after drying, whereas Synechocystis sp. PCC 6803 survived the coating formation, as shown by an adenosine triphosphate (ATP) assay. Synechocystis sp. PCC 6803 consumed oxygen (by cell respiration) for up to 5 days, but was unable to perform photosynthesis, as indicated by a lack of oxygen evolution. However, Chroococcidiopsis cubana PCC 7433, a strain of desiccation-resistant extremophilic cyanobacteria, survived and performed photosynthesis and carbon capture within the biocoating, with specific rates of oxygen evolution up to 0.4 g of oxygen/g of biomass per day. Continuous measurements of dissolved oxygen were carried out over a month and showed no sign of decreasing activity. Extremophilic cyanobacteria are viable in a variety of environments, making them ideal candidates for use in biocoatings and other biotechnology. IMPORTANCE As water has become a precious resource, there is a growing need for less water-intensive use of microorganisms, while avoiding desiccation stress. Mechanically robust, ready-to-use biocoatings or "living paints" (a type of artificial biofilm consisting of a synthetic matrix containing functional bacteria) represent a novel way to address these issues. Here, we describe the revolutionary, first-ever use of an extremophilic cyanobacterium (Chroococcidiopsis cubana PCC 7433) in biocoatings, which were able to produce high levels of oxygen and carbon capture for at least 1 month despite complete desiccation and subsequent rehydration. Beyond culturing viable bacteria with reduced water resources, this pioneering use of extremophiles in biocoatings could be further developed for a variety of applications, including carbon capture, wastewater treatment and biofuel production.

Dysregulated NAD(H) homeostasis associated with ciprofloxacin tolerance in Escherichia coli investigated on a single-cell level with the Peredox [NADH:NAD+] biosensor.

Introduction: Antibiotic persistence (subpopulation tolerance) occurs when a subpopulation of antibiotic sensitive cells survives prolonged exposure to a bactericidal concentration of an antibiotic, and is capable of regrowth once the antibiotic is removed. This phenomenon has been shown to contribute to prolonged treatment duration, infection recurrence, and accelerated development of genetic resistance. Currently, there are no biomarkers which would allow for segregation of these antibiotic-tolerant cells from the bulk population prior to antibiotic exposure, limiting research on this phenomenon to retrograde analyses. However, it has been previously shown that persisters often have a dysregulated intracellular redox homeostasis, warranting its investigation as a potential marker for antibiotic tolerance. Furthermore, it is currently unknown whether another antibiotic tolerant subpopulation - viable but non-culturable cells (VBNCs), are simply persisters with extreme lag phase, or are formed through separate pathways. VBNCs similarly to persisters remain viable following antibiotic exposure, however, are not capable of regrowth in standard conditions. Methods: In this article we employed an NADH:NAD+ biosensor (Peredox) to investigate NADH homeostasis of ciprofloxacin-tolerant E. coli cells on a single-cell level. [NADH:NAD+] was used as a proxy for measuring intracellular redox homeostasis and respiration rate. Results and Discussion: First, we demonstrated that ciprofloxacin exposure results in a high number of VBNCs, several orders of magnitude higher than persisters. However, we found no correlation in the frequencies of persister and VBNC subpopulations. Ciprofloxacin-tolerant cells (persisters & VBNCs) were actively undergoing respiration, although at a significantly lower rate on average when compared to the bulk population. We also noted significant heterogeneity on a single-cell level within the subpopulations, however were unable to segregate persisters from VBNCs based on these observations alone. Finally, we showed that in the highly-persistent strain of E. coli, E. coli HipQ, ciprofloxacin-tolerant cells have a significantly lower [NADH:NAD+] ratio than tolerant cells of its parental strain, providing further link between disturbed NADH homeostasis and antibiotic tolerance.

Solid waste motor tricycle operators in Kumasi, Ghana, harbour respiratory pathogens; a public health threat.

BACKGROUND: The use of motor tricycles in transporting municipal solid waste (MSW) within urban and peri-urban towns in Ghana is on the increase. This activity often leads to the introduction of pathogen-containing bioaerosols into the environment, as well as to the tricycle operators. We sought to investigate the prevalence and associated risk factors of respiratory pathogens among solid waste tricycle operators. METHODS: A cross-sectional study was conducted among 155 solid waste transporters who use motor tricycles using semi-structured interviews. Nasopharyngeal swabs were obtained from participants and screened for respiratory pathogens using Polymerase Chain Reaction (PCR). RESULTS: Pathogens detected in participants were SARS-CoV-2 (n = 10, 6.5%) and Streptococcus pneumoniae (n = 10, 6.5%), constituting an overall prevalence of 12.9% and co-infection rate of 1.3%. The most common self-reported symptoms were cough (n = 67, 43.2%), sore throat (n = 44, 28.4%) and difficulty in breathing (n = 22, 14.2%). Adherence to the use of gloves (n = 117, 75.5%) and nose mask (n = 110, 71.0%) was high. There was a significant association between the detection of respiratory pathogens and the use of gloves, use of more than one PPE and exposure to other pollutants (p < 0.05). Individuals who were exposed to "other pollutants" significantly had lower odds of becoming infected with respiratory pathogens (Adj. OR (95% CI): 0.119(0.015,0.938). CONCLUSION: Although prevalence of respiratory pathogens is generally low, strict adherence to PPE use could further reduce its rates to even lower levels. Governmental health institutions and informal solid waste transporters should address challenges related to exposure to pollutants, use of gloves, and multiple PPE.

A multiplex PCR assay for the differentiation of Mycobacterium tuberculosis complex reveals high rates of mixed-lineage tuberculosis infections among patients in Ghana.

In low-resource settings with high tuberculosis (TB) burdens, lack of rapid diagnostic methods for detection and differentiation of Mycobacterium tuberculosis complex (MTBC) is a major challenge affecting TB management. This study utilized comparative genomic analyses of MTBC lineages; M. tuberculosis, M. africanum Lineages 5/6 and M. bovis to identify lineage-specific genes. Primers were designed for the development of a Multiplex PCR assay which was successful in differentiating the MTBC lineages. There was no cross-reaction with other respiratory pathogens tested. Validation of the assay using clinical samples was performed with sputum DNA extracts from 341 clinically confirmed active TB patients. It was observed that 24.9% of cases were caused by M. tuberculosis, while M. africanum L5 & L6 reported 9.0% and 14.4%, respectively. M. bovis infection was the least frequently detected lineage with 1.8%. Also, 27.0% and 17.0% of the cases were PCR negative and unspeciated, respectively. However, mixed-lineage TB infections were recorded at a surprising 5.9%. This multiplex PCR assay will allow speciation of MTBC lineages in low-resource regions, providing rapid differentiation of TB infections to select appropriate medication at the earliest possible time point. It will also be useful in epidemiological surveillance studies providing reliable information on the prevalence of TB lineages as well as identifying difficult to treat cases of mixed-lineage tuberculosis infections.

The Use of Comparative Genomic Analysis for the Development of Subspecies-Specific PCR Assays for Mycobacterium abscessus.

Mycobacterium abscessus complex (MABC) is an important pathogen of immunocompromised patients. Accurate and rapid determination of MABC at the subspecies level is vital for optimal antibiotic therapy. Here we have used comparative genomics to design MABC subspecies-specific PCR assays. Analysis of single nucleotide polymorphisms and core genome multilocus sequence typing showed clustering of genomes into three distinct clusters representing the MABC subspecies M. abscessus, M. bolletii and M. massiliense. Pangenome analysis of 318 MABC genomes from the three subspecies allowed for the identification of 15 MABC subspecies-specific genes. In silico testing of primer sets against 1,663 publicly available MABC genomes and 66 other closely related Mycobacterium genomes showed that all assays had >97% sensitivity and >98% specificity. Subsequent experimental validation of two subspecies-specific genes each showed the PCR assays worked well in individual and multiplex format with no false-positivity with 5 other mycobacteria of clinical importance. In conclusion, we have developed a rapid, accurate, multiplex PCR-assay for discriminating MABC subspecies that could improve their detection, diagnosis and inform correct treatment choice.

Introducing Porosity in Colloidal Biocoatings to Increase Bacterial Viability.

A biocoating confines nongrowing, metabolically active bacteria within a synthetic colloidal polymer (i.e., latex) film. Bacteria encapsulated inside biocoatings can perform useful functions, such as a biocatalyst in wastewater treatment. A biocoating needs to have a high permeability to allow a high rate of mass transfer for rehydration and the transport of both nutrients and metabolic products. It therefore requires an interconnected porous structure. Tuning the porosity architecture is a challenge. Here, we exploited rigid tubular nanoclays (halloysite) and nontoxic latex particles (with a relatively high glass transition temperature) as the colloidal "building blocks" to tailor the porosity inside biocoatings containing Escherichia coli bacteria as a model organism. Electron microscope images revealed inefficient packing of the rigid nanotubes and proved the existence of nanovoids along the halloysite/polymer interfaces. Single-cell observations using confocal laser scanning microscopy provided evidence for metabolic activity of the E. coli within the biocoatings through the expression of a yellow fluorescent protein. A custom-built apparatus was used to measure the permeability of a fluorescein sodium salt in the biocoatings. Whereas there was no measurable permeability in a coating made from only latex particles, the permeability coefficient of the composite biocoatings increased with increasing halloysite content up to a value of 1 × 10-4 m h-1. The effects of this increase in permeability was demonstrated through a specially developed resazurin reduction assay. Bacteria encapsulated in halloysite composite biocoatings had statistically significant higher metabolic activities in comparison to bacteria encapsulated in a nonoptimized coating made from latex particles alone.

Loss of phenotypic inheritance associated with ydcI mutation leads to increased frequency of small, slow persisters in Escherichia coli.

Whenever a genetically homogenous population of bacterial cells is exposed to antibiotics, a tiny fraction of cells survives the treatment, the phenomenon known as bacterial persistence [G.L. Hobby et al., Exp. Biol. Med. 50, 281-285 (1942); J. Bigger, The Lancet 244, 497-500 (1944)]. Despite its biomedical relevance, the origin of the phenomenon is still unknown, and as a rare, phenotypically resistant subpopulation, persisters are notoriously hard to study and define. Using computerized tracking we show that persisters are small at birth and slowly replicating. We also determine that the high-persister mutant strain of Escherichia coli, HipQ, is associated with the phenotype of reduced phenotypic inheritance (RPI). We identify the gene responsible for RPI, ydcI, which encodes a transcription factor, and propose a mechanism whereby loss of phenotypic inheritance causes increased frequency of persisters. These results provide insight into the generation and maintenance of phenotypic variation and provide potential targets for the development of therapeutic strategies that tackle persistence in bacterial infections.

Trajectory energy minimization for cell growth tracking and genealogy analysis.

Cell growth experiments with a microfluidic device produce large-scale time-lapse image data, which contain important information on cell growth and patterns in their genealogy. To extract such information, we propose a scheme to segment and track bacterial cells automatically. In contrast with most published approaches, which often split segmentation and tracking into two independent procedures, we focus on designing an algorithm that describes cell properties evolving between consecutive frames by feeding segmentation and tracking results from one frame to the next one. The cell boundaries are extracted by minimizing the distance regularized level set evolution (DRLSE) model. Each individual cell was identified and tracked by identifying cell septum and membrane as well as developing a trajectory energy minimization function along time-lapse series. Experiments show that by applying this scheme, cell growth and division can be measured automatically. The results show the efficiency of the approach when testing on different datasets while comparing with other existing algorithms. The proposed approach demonstrates great potential for large-scale bacterial cell growth analysis.

ESX1-dependent fractalkine mediates chemotaxis and Mycobacterium tuberculosis infection in humans.

Mycobacterium tuberculosis-induced cellular aggregation is essential for granuloma formation and may assist establishment and early spread of M. tuberculosis infection. The M. tuberculosis ESX1 mutant, which has a non-functional type VII secretion system, induced significantly less production of the host macrophage-derived chemokine fractalkine (CX3CL1). Upon infection of human macrophages ESX1-dependent fractalkine production mediated selective recruitment of CD11b+ monocytic cells and increased infection of neighbouring cells consistent with early local spread of infection. Fractalkine levels were raised in vivo at tuberculous disease sites in humans and were significantly associated with increased CD11b+ monocytic cellular recruitment and extent of granulomatous disease. These findings suggest a novel fractalkine-dependent ESX1-mediated mechanism in early tuberculous disease pathogenesis in humans. Modulation of M. tuberculosis-mediated fractalkine induction may represent a potential treatment option in the future, perhaps allowing us to switch off a key mechanism required by the pathogen to spread between cells.

Undetected multidrug-resistant tuberculosis amplified by first-line therapy in mixed infection.

Infections with >1 Mycobacterium tuberculosis strain(s) are underrecognized. We show, in vitro and in vivo, how first-line treatment conferred a competitive growth advantage to amplify a multidrug-resistant M. tuberculosis strain in a patient with mixed infection. Diagnostic techniques that identify mixed tubercle bacilli populations are needed to curb the spread of multidrug resistance.

The dormancy regulator DosR controls ribosome stability in hypoxic mycobacteria.

It is thought that during latent infection, Mycobacterium tuberculosis bacilli are retained within granulomas in a low-oxygen environment. The dormancy survival (Dos) regulon, regulated by the response regulator DosR, appears to be essential for hypoxic survival in M. tuberculosis, but it is not known how the regulon promotes survival. Here we report that mycobacteria, in contrast to enteric bacteria, do not form higher-order structures (e.g. ribosomal dimers) upon entry into stasis. Instead, ribosomes are stabilized in the associated form (70S). Using a strategy incorporating microfluidic, proteomic, and ribosomal profiling techniques to elucidate the fate of mycobacterial ribosomes during hypoxic stasis, we show that the dormancy regulator DosR is required for optimal ribosome stabilization. We present evidence that the majority of this effect is mediated by the DosR-regulated protein MSMEG_3935 (a S30AE domain protein), which is associated with the ribosome under hypoxic conditions. A Δ3935 mutant phenocopies the ΔdosR mutant during hypoxia, and complementation of ΔdosR with the MSMEG_3935 gene leads to complete recovery of dosR mutant phenotypes during hypoxia. We suggest that this protein is named ribosome-associated factor under hypoxia (RafH) and that it is the major factor responsible for DosR-mediated hypoxic survival in mycobacteria.

Rv3615c is a highly immunodominant RD1 (Region of Difference 1)-dependent secreted antigen specific for Mycobacterium tuberculosis infection.

The 6-kDa early secretory antigenic target of Mycobacterium tuberculosis (ESAT-6) and the 10-kDa culture filtrate antigen (CFP-10), encoded in region of difference 1 (RD1) and secreted by the ESAT-6 system 1 (Esx-1) secretion system, are the most immunodominant and highly M. tuberculosis (MTB)-specific antigens. These attributes are responsible for their primary importance in tuberculosis (TB) immunodiagnosis and vaccine development. Rv3615c [Esx-1 substrate protein C (EspC)], encoded outside RD1, is similar in size and sequence homology to CFP-10 and ESAT-6, suggesting it might be a target of cellular immunity in TB. Using ex vivo enzyme-linked immunospot- and flow cytometry-based cytokine-secretion assay, we comprehensively assessed cellular immune responses to EspC in patients with active TB, latently infected persons, and uninfected bacillus Calmette-Guérin (BCG)-vaccinated controls. EspC was at least as immunodominant as ESAT-6 and CFP-10 in both active and latent TB infection. EspC contained broadly recognized CD4(+) and CD8(+) epitopes, inducing a predominantly CD4(+) T-cell response that comprised functional T-cell subsets secreting both IFN-γ and IL-2 as well as functional T-cell subsets secreting only IFN-γ. Surprisingly, T-cell responses to EspC were as highly specific (93%) for MTB infection as responses to ESAT-6 and CFP-10, with only 2 of 27 BCG-vaccinated controls responding to each antigen. Using quantitative proteomics and metabolically labeled mutant and genetically complemented MTB strains, we identified the mechanism of the specificity of anti-EspC immunity as the Esx-1 dependence of EspC secretion. The high immunodominance of EspC, equivalent to that of ESAT-6 and CFP-10, makes it a TB vaccine candidate, and its high specificity confers strong potential for T-cell-based immunodiagnosis.

The primary mechanism of attenuation of bacillus Calmette-Guerin is a loss of secreted lytic function required for invasion of lung interstitial tissue.

Tuberculosis remains a leading cause of death worldwide, despite the availability of effective chemotherapy and a vaccine. Bacillus Calmette-Guérin (BCG), the tuberculosis vaccine, is an attenuated mutant of Mycobacterium bovis that was isolated after serial subcultures, yet the functional basis for this attenuation has never been elucidated. A single region (RD1), which is absent in all BCG substrains, was deleted from virulent M. bovis and Mycobacterium tuberculosis strains, and the resulting DeltaRD1 mutants were significantly attenuated for virulence in both immunocompromised and immunocompetent mice. The M. tuberculosis DeltaRD1 mutants were also shown to protect mice against aerosol challenge, in a similar manner to BCG. Interestingly, the DeltaRD1 mutants failed to cause cytolysis of pneumocytes, a phenotype that had been previously used to distinguish virulent M. tuberculosis from BCG. A specific transposon mutation, which disrupts the Rv3874 Rv3875 (cfp-10 esat-6) operon of RD1, also caused loss of the cytolytic phenotype in both pneumocytes and macrophages. This mutation resulted in the attenuation of virulence in mice, as the result of reduced tissue invasiveness. Moreover, specific deletion of each transcriptional unit of RD1 revealed that three independent transcriptional units are required for virulence, two of which are involved in the secretion of ESAT-6 (6-kDa early secretory antigenic target). We conclude that the primary attenuating mechanism of bacillus Calmette-Guérin is the loss of cytolytic activity mediated by secreted ESAT-6, which results in reduced tissue invasiveness.

Survival perspectives from the world's most successful pathogen, Mycobacterium tuberculosis.

Studying defined mutants of Mycobacterium tuberculosis in the mouse model of infection has led to the discovery of attenuated mutants that fall into several phenotypic classes. These mutants are categorized by their growth characteristics compared with those of wild-type M. tuberculosis, and include severe growth in vivo mutants, growth in vivo mutants, persistence mutants, pathology mutants and dissemination mutants. Here, examples of each of these mutant phenotypes are described and classified accordingly. Defining the importance of mycobacterial gene products responsible for in vivo growth, persistence and the induction of immunopathology will lead to a greater understanding of the host-pathogen interaction and potentially to new antimycobacterial treatment options.

Survival of Mycobacterium tuberculosis in host macrophages involves resistance to apoptosis dependent upon induction of antiapoptotic Bcl-2 family member Mcl-1.

Mcl-1 protein expression was found to be up-regulated during infection with virulent Mycobacterium tuberculosis strain H37Rv. Mcl-1 induction in THP-1 cells was optimal at a multiplicity of infection of 0.8-1.2 bacilli per macrophage and was independent of opsonin coating of the bacteria. Mcl-1 expression was elevated as early as 4 h, peaked at 5.8-fold above control cells at 24 h, and remained elevated at 48 h after infection. In THP-1 cells, mMcl-1 mRNA was induced by infection with live H37Rv but not with attenuated M. tuberculosis strain H37Ra, heat-killed H37Rv, or latex beads. In THP-1 cells and monocyte-derived macrophages (MDMs), Mcl-1 protein was induced by infection with live H37Rv but not with attenuated M. tuberculosis strain H37Ra, heat-killed H37Rv, or latex beads. Treatment of uninfected, H37Ra-infected, and H37Rv-infected THP-1 cells and MDMs with antisense oligonucleotides to mcl-1 reduced Mcl-1 expression by >84%. This resulted in an increase in apoptosis of both MDMs and THP-1 cells that were infected with H37Rv, but not cells that were uninfected or infected with H37Ra. Increased apoptosis correlated with a decrease in M. tuberculosis CFUs recovered from antisense-treated, H37Rv-infected cells at 4 and 7 days after infection. In contrast, CFU recoveries from sense-treated, H37Rv-infected cells or from antisense- or sense-treated, H37Ra-infected cells were unchanged from controls. Thus, the antiapoptotic effect of the induction of Mcl-1 expression in H37Rv-infected macrophages promotes the survival of virulent M. tuberculosis.