A microfluidic cell-trapping device to study dynamic host-microbe interactions at the single-cell level.

Single-cell imaging of host-microbe interactions over time is impeded by cellular motility because the cells under scrutiny tend to migrate out of the imaging field. To overcome this technical challenge, we developed a microfluidic platform for imaging hundreds of individual motile phagocytic cells and bacteria within microfluidic traps that restrict their movement. The interaction of trapped host cells and bacteria is monitored by long-term time-lapse microscopy, allowing direct visualization of all stages of infection at the single-cell level. The medium flowing through the microfluidic device can be changed quickly and precisely, permitting the real-time imaging of cellular responses to antibiotics or other environmental stresses. Here, we demonstrate the potential applications of this approach by co-culturing the phagocytic amoeba Dictyostelium discoideum with the intracellular pathogen Mycobacterium marinum. However, the platform can be adapted easily for use with other host cells or microorganisms. This approach will provide new insights into host-pathogen interactions that cannot be studied using conventional population-based assays.

A microfluidic cell-trapping device for single-cell tracking of host-microbe interactions.

The impact of cellular individuality on host-microbe interactions is increasingly appreciated but studying the temporal dynamics of single-cell behavior in this context remains technically challenging. Here we present a microfluidic platform, InfectChip, to trap motile infected cells for high-resolution time-lapse microscopy. This approach allows the direct visualization of all stages of infection, from bacterial uptake to death of the bacterium or host cell, over extended periods of time. We demonstrate the utility of this approach by co-culturing an established host-cell model, Dictyostelium discoideum, with the extracellular pathogen Klebsiella pneumoniae or the intracellular pathogen Mycobacterium marinum. We show that the outcome of such infections is surprisingly heterogeneous, ranging from abortive infection to death of the bacterium or host cell. InfectChip thus provides a simple method to dissect the time-course of host-microbe interactions at the single-cell level, yielding new insights that could not be gleaned from conventional population-based measurements.

The FBPase Encoding Gene glpX Is Required for Gluconeogenesis, Bacterial Proliferation and Division In Vivo of Mycobacterium marinum.

Lipids have been identified as important carbon sources for Mycobacterium tuberculosis (Mtb) to utilize in vivo. Thus gluconeogenesis bears a key role for Mtb to survive and replicate in host. A rate-limiting enzyme of gluconeogenesis, fructose 1, 6-bisphosphatase (FBPase) is encoded by the gene glpX. The functions of glpX were studied in M. marinum, a closely related species to Mtb. The glpX deletion strain (ΔglpX) displayed altered gluconeogenesis, attenuated virulence, and altered bacterial proliferation. Metabolic profiles indicate an accumulation of the FBPase substrate, fructose 1, 6-bisphosphate (FBP) and altered gluconeogenic flux when ΔglpX is cultivated in a gluconeogenic carbon substrate, acetate. In both macrophages and zebrafish, the proliferation of ΔglpX was halted, resulting in dramatically attenuated virulence. Intracellular ΔglpX exhibited an elongated morphology, which was also observed when ΔglpX was grown in a gluconeogenic carbon source. This elongated morphology is also supported by the observation of unseparated multi-nucleoid cell, indicating that a complete mycobacterial division in vivo is correlated with intact gluconeogenesis. Together, our results indicate that glpX has essential functions in gluconeogenesis, and plays an indispensable role in bacterial proliferation in vivo and virulence of M. marinum.

Electromechanical cell lysis using a portable audio device: enabling challenging sample preparation at the point-of-care.

Audio sources are ubiquitously available on portable electronic devices, including cell phones. Here we demonstrate lysis of Mycobacterium marinum and Staphylococcus epidermidis bacteria utilizing a portable audio device coupled with a simple and inexpensive electromagnetic coil. The resulting alternating magnetic field rotates a magnet in a tube with the sample and glass beads, lysing the cells and enabling sample preparation for these bacteria anywhere there is a cell phone, mp3 player, laptop, or other device with a headphone jack.

Direct detection of bacterial protein secretion using whole colony proteomics.

Bacteria use a variety of secretion systems to transport proteins beyond their cell membrane to interact with their environment. For bacterial pathogens, these systems are key virulence determinants that transport bacterial proteins into host cells. Genetic screens to identify bacterial genes required for export have relied on enzymatic or fluorescent reporters fused to known substrates to monitor secretion. However, they cannot be used in analysis of all secretion systems, limiting the implementation across bacteria. Here, we introduce the first application of a modified form of whole colony MALDI-TOF MS to directly detect protein secretion from intact bacterial colonies. We show that this method is able to specifically monitor the ESX-1 system protein secretion system, a major virulence determinant in both mycobacterial and Gram-positive pathogens that is refractory to reporter analysis. We validate the use of this technology as a high throughput screening tool by identifying an ESAT-6 system 1-deficient mutant from a Mycobacterium marinum transposon insertion library. Furthermore, we also demonstrate detection of secreted proteins of the prevalent type III secretion system from the Gram-negative pathogen, Pseudomonas aeruginosa. This method will be broadly applicable to study other bacterial protein export systems and for the identification of compounds that inhibit bacterial protein secretion.

Phenotypic and genotypic characteristics of Mycobacterium isolates from fighting fish Betta spp. in Malaysia.

Mycobacteriosis due to mycobacteria is one of the most common bacterial diseases in ornamental fish. We describe here the phenotypic and genotypic characteristics of Mycobacterium isolates from fighting fish Betta spp. using ATCC Mycobacterium marinum, Mycobacterium fortuitum and Mycobacterium chelonae as references. A total of four isolates (M1, M2, M3, M4) were obtained from four out of 106 fish samples using selective agar, and identified to Mycobacterium genus using acid-fast staining and 16s rRNA gene-based genus specific polymerase chain reaction. DNA sequencing and NCBI-BLAST analysis further identified isolate M1 as M. marinum and isolates M2, M3, M4 as M. fortuitum. Morphological, physiological and biochemical tests were carried out for phenotypic characterizations. Universal M13 and wild-type phage M13 RAPD dendogram was generated to illustrate the genetic relationship of the isolates and reference strains.

Infección por Mycobacterium marinum. Presentación de un nuevo caso y revisión de la literatura / Mycobacterium marinuminfection. Case report and review of the literature

Las infecciones por micobacterias atípicas están adquiriendo cada vez mayor importancia en los pacientes inmunodeprimidos, así como en huéspedes sanos. El Mycobacterium marinum es la micobacteria atípica que afecta la piel con mayor frecuencia. Debe sospecharse ante la existencia de úlceras, nódulos o placas crónicas y el antecedente de un contacto con medios acuáticos. El tratamiento óptimo no está aún bien establecido. Presentamos un nuevo caso de infección por Mycobacterium marinum en un paciente que seguía tratamiento con fármacos inmunosupresores, que respondió favorablemente al tratamiento con doxiciclina y revisamos los distintos regímenes antibióticos utilizados para el tratamiento de la infección por Mycobacterium marinum en los últimos años

Atypical mycobacterial infections are increasingly important in immunosuppressed patients as well as in healthy hosts. The atypical mycobacterium that most commonly affects the skin is Mycobacterium marinum. The infection should be suspected upon the presence of ulcers, nodules or chronic plaques and a history of contact with fresh or salt water. Optimal therapy is yet to be established. We report a case of Mycobacterium marinum infection in a patient receiving immunosuppressive therapy that responded favourably to treatment with doxicycline. We review the different antibiotic regimens prescribed in the past years for the treatment of Mycobacterium marinum infection

Mycobacterium marinum biofilm formation reveals cording morphology.

Abstract The emergence of the nontuberculosis mycobacteria (NTM) as clinically relevant pathogens has warranted the study of these ubiquitous organisms in the context of their likely environmental niche, the biofilm. We assayed the NTM bacterium Mycobacterium marinum strain 1218R, a fish outbreak isolate, for biofilm formation on different surfaces over time using three different methods. Using the MBEC system, biofilm development occurred continually over the 14-day culture period reaching a mature or stable biofilm state after 7 days postinoculation. Quantification of M. marinum biofilm formation on high-density polyethylene (HDPE), polycarbonate (PC) and silicon (Si) coupons over a 14-day period was evaluated using a continuous flow reactor system. M. marinum developed biofilms on all of the surfaces tested. However, substantially more biofilm accumulated on the silicon than on the other substrates (Si>HDPE>PC) under the same growth conditions indicating that silicon was the most effective substratum studied for the generation of M. marinum biofilms and suggesting a correlation between surface hydrophobicity and attachment. Finally, confocal laser scanning microscopy was used to visualize M. marinum biofilm development in situ over time and revealed an unusual biofilm ultrastructure. Large cell clusters attached to the surface grew in parallel sinuous arrays of cells that formed large cords.