Exposure to a fungicide for a field-realistic duration does not alter bumble bee fecal microbiota structure.

Social bees are frequently exposed to pesticides when foraging on nectar and pollen. Recent research has shown that pesticide exposure not only impacts social bee host health but can also alter the community structure of social bee gut microbiotas. However, most research on pesticide-bee gut microbiota interactions has been conducted in honey bees; bumble bees, native North American pollinators, have received less attention and, due to differences in their ecology, may be exposed to certain pesticides for shorter durations than honey bees. Here, we examine how exposure to the fungicide chlorothalonil for a short, field-realistic duration alters bumble bee fecal microbiotas (used as a proxy for gut microbiotas) and host performance. We expose small groups of Bombus impatiens workers (microcolonies) to field-realistic chlorothalonil concentrations for 5 days, track changes in fecal microbiotas during the exposure period and a recovery period, and compare microcolony offspring production between treatments at the end of the experiment. We also assess the use of fecal microbiotas as a gut microbiota proxy by comparing community structures of fecal and gut microbiotas. We find that chlorothalonil exposure for a short duration does not alter bumble bee fecal microbiota structure or affect microcolony production at any concentration but that fecal and gut microbiotas differ significantly in community structure. Our results show that, at least when exposure durations are brief and unaccompanied by other stressors, bumble bee microbiotas are resilient to fungicide exposure. Additionally, our work highlights the importance of sampling gut microbiotas directly, when possible.IMPORTANCEWith global pesticide use expected to increase in the coming decades, studies on how pesticides affect the health and performance of animals, including and perhaps especially pollinators, will be crucial to minimize negative environmental impacts of pesticides in agriculture. Here, we find no effect of exposure to chlorothalonil for a short, field-realistic period on bumble bee fecal microbiota community structure or microcolony production regardless of pesticide concentration. Our results can help inform pesticide use practices to minimize negative environmental impacts on the health and fitness of bumble bees, which are key native, commercial pollinators in North America. We also find that concurrently sampled bumble bee fecal and gut microbiotas contain similar microbes but differ from one another in community structure and consequently suggest that using fecal microbiotas as a proxy for gut microbiotas be done cautiously; this result contributes to our understanding of proxy use in gut microbiota research.

A study on twitching motility dynamics in Ralstonia solanacearum microcolonies by live imaging.

Ralstonia solanacearum is a rod-shaped phytopathogenic bacterium that causes lethal wilt disease in many plants. On solid agar growth medium, in the early hour of the growth of the bacterial colony, the type IV pili-mediated twitching motility, which is important for its virulence and biofilm formation, is prominently observed under the microscope. In this study, we have done a detailed observation of twitching motility in R. solanacearum colony. In the beginning, twitching motility in the microcolonies was observed as a density-dependent phenomenon that influences the shape of the microcolonies. No such phenomenon was observed in Escherichia coli, where twitching motility is absent. In the early phase of colony growth, twitching motility exhibited by the cells at the peripheral region of the colony was more prominent than the cells toward the center of the colony. Using time-lapse photography and merging the obtained photomicrographs into a video, twitching motility was observed as an intermittent phenomenon that progresses in layers in all directions as finger-like projections at the peripheral region of a bacterial colony. Each layer of bacteria twitches on top of the other and produces a multilayered film-like appearance. We found that the duration between the emergence of each layer diminishes progressively as the colony becomes older. This study on twitching motility demonstrates distinctly heterogeneity among the cells within a colony regarding their dynamics and the influence of microcolonies on each other regarding their morphology.

What's in a name? Characteristics of clinical biofilms.

In vitro biofilms are communities of microbes with unique features compared to individual cells. Biofilms are commonly characterized by physical traits like size, adhesion, and a matrix made of extracellular substances. They display distinct phenotypic features, such as metabolic activity and antibiotic tolerance. However, the relative importance of these traits depends on the environment and bacterial species. Various mechanisms enable biofilm-associated bacteria to withstand antibiotics, including physical barriers, physiological adaptations, and changes in gene expression. Gene expression profiles in biofilms differ from individual cells but, there is little consensus among studies and so far, a 'biofilm signature transcriptome' has not been recognized. Additionally, the spatial and temporal variability within biofilms varies greatly depending on the system or environment. Despite all these variable conditions, which produce very diverse structures, they are all noted as biofilms. We discuss that clinical biofilms may differ from those grown in laboratories and found in the environment and discuss whether the characteristics that are commonly used to define and characterize biofilms have been shown in infectious biofilms. We emphasize that there is a need for a comprehensive understanding of the specific traits that are used to define bacteria in infections as clinical biofilms.

Mechanisms of Long-Term Persistence of Mycoplasmas in Children with Asthma.

Improving the management of children with asthma associated with mycoplasma infection is important. Aim: To study the duration of the persistence of antigens, and DNA in a free state, in the structures of circulating immune complexes (CICs) and living cells of Mycoplasma pneumoniae (Mpn) and Mycoplasma hominis (Mh) in children with asthma. In total, 205 children with asthma from 1 to 14 years were observed. The reaction of aggregate-hemagglutination (AHAA), the direct immunofluorescence reaction (DIF), the reaction of the polymerase chain reaction (PCR), and the culture method were used. In addition, 47 children were re-examined 1.5 months after the treatment of mycoplasma infection with azithromycin. The number of samples positive for antigens and DNA in the free state and in the structures of CICs significantly decreased. Then, 50 blood serum samples containing Mh antigens, and 50 samples containing Mpn antigens were analyzed by culture method. Mh was isolated in 21 (65.5%) of 32 samples containing DNA. Mpn was isolated from antigen-positive samples in nine cases. The presented data indicate the long-term persistence of antigens, and DNA of mycoplasma cells in the free state, in the structure of CICs, as well as in the form of "microcolonies". A high level of CICs can be used to predict the course of the disease and the response to therapy.

Perception, regulation, and fitness effects of pollen phytosterols in the bumble bee, Bombus terrestris.

PREMISE: Many flowering plants depend on insects for pollination and thus attract pollinators by offering rewards, mostly nectar and pollen. Bee pollinators rely on pollen as their main nutrient source. Pollen provides all essential micro- and macronutrients including substances that cannot be synthesized by bees themselves, such as sterols, which bees need for processes such as hormone production. Variations in sterol concentrations may consequently affect bee health and reproductive fitness. We therefore hypothesized that (1) these variations in pollen sterols affect longevity and reproduction in bumble bees and (2) can thus be perceived via the bees' antennae before consumption. METHODS: We studied the effect of sterols on longevity and reproduction of Bombus terrestris workers in feeding experiments and investigated sterol perception using chemotactile proboscis extension response (PER) conditioning. RESULTS: Workers could perceive several sterols (cholesterol, cholestenone, desmosterol, stigmasterol, ß-sitosterol) via their antennae but not differentiate between them. However, when sterols were presented in pollen, and not as a single compound, the bees were unable to differentiate between pollen differing in sterol content. Additionally, different sterol concentrations in pollen neither affected pollen consumption nor brood development or worker longevity. CONCLUSIONS: Since we used both natural concentrations and concentrations higher than those found in pollen, our results indicate that bumble bees may not need to pay specific attention to pollen sterol content beyond a specific threshold. Naturally encountered concentrations might fully support their sterol requirements and higher concentrations do not seem to have negative effects.

Analysis of the Adhesive-Invasive Potential of Two Morphologically Different Types of Mycoplasma hominis Colonies.

Mycoplasma hominis is an opportunistic human pathogen that causes acute and chronic infections of the urogenital tract. A new form of M. hominis colonies (microcolonies) was isolated, that differed from typical colonies by morphology, size, growth rate, and resistance to unfavorable factors, in particular, to antibiotics. The formation of microcolonies is associated with a switch in energy metabolism towards nucleoside utilization, which leads to a decrease in energy production and a transition to a persistor-like state. Typical and microcolony cultures of M. hominis H-34 were obtained and a comparative analysis of their adhesive-invasive potential, morphology, and size was carried out. It was shown that both typical and microcolonies can effectively attach and penetrate into HeLa cells. Unlike microcolonies, the morphology and size of cells in typical colonies change significantly after HeLa infection. This indicates functional changes in cells of typical colonies during infection.

A Role for the RNA Polymerase Gene Specificity Factor σ54 in the Uniform Colony Growth of Uropathogenic Escherichia coli.

The canonical function of a bacterial sigma (σ) factor is to determine the gene specificity of the RNA polymerase (RNAP). In several diverse bacterial species, the σ54 factor uniquely confers distinct functional and regulatory properties on the RNAP. A hallmark feature of the σ54-RNAP is the obligatory requirement for an activator ATPase to allow transcription initiation. Different activator ATPases couple diverse environmental cues to the σ54-RNAP to mediate adaptive changes in gene expression. Hence, the genes that rely upon σ54 for their transcription have a wide range of different functions suggesting that the repertoire of functions performed by genes, directly or indirectly affected by σ54, is not yet exhaustive. By comparing the growth patterns of prototypical enteropathogenic, uropathogenic, and nonpathogenic Escherichia coli strains devoid of σ54, we uncovered that the absence of σ54 results in two differently sized colonies that appear at different times specifically in the uropathogenic E. coli (UPEC) strain. Notably, UPEC bacteria devoid of individual activator ATPases of the σ54-RNAP do not phenocopy the σ54 mutant strain. Thus, it seems that σ54's role as a determinant of uniform colony appearance in UPEC bacteria represents a putative non-canonical function of σ54 in regulating genetic information flow. IMPORTANCE RNA synthesis is the first step of gene expression. The multisubunit RNA polymerase (RNAP) is the central enzyme responsible for RNA synthesis in bacteria. The dissociable sigma (σ) factor subunit directs the RNAP to different sets of genes to allow their expression in response to various cellular needs. Of the seven σ factors in Escherichia coli and related bacteria, σ54 exists in a class of its own. This study has uncovered that σ54 is a determinant of the uniform growth of uropathogenic E. coli on solid media. This finding suggests a role for this σ54 in gene regulation that extends beyond its known function as an RNAP gene specificity factor.

Advanced Understanding of Prokaryotic Biofilm Formation through Use of a Cost-Effective and Versatile Multipanel Adhesion (mPAD) Mount.

Most microorganisms exist in biofilms, which comprise aggregates of cells surrounded by an extracellular matrix that provides protection from external stresses. Based on the conditions under which they form, biofilm structures vary in significant ways. For instance, biofilms that develop when microbes are incubated under static conditions differ from those formed when microbes encounter the shear forces of a flowing liquid. Moreover, biofilms develop dynamically over time. Here, we describe a cost-effective coverslip holder, printed with a three-dimensional (3D) printer, that facilitates surface adhesion assays under a broad range of standing and shaking culture conditions. This multipanel adhesion (mPAD) mount further allows cultures to be sampled at multiple time points, ensuring consistency and comparability between samples and enabling analyses of the dynamics of biofilm formation. As a proof of principle, using the mPAD mount for shaking, oxic cultures, we confirm previous flow chamber experiments showing that the Pseudomonas aeruginosa wild-type strain and a phenazine deletion mutant (Δphz) strain form biofilms with similar structure but reduced density in the mutant strain. Extending this analysis to anoxic conditions, we reveal that microcolony formation and biofilm formation can only be observed under shaking conditions and are decreased in the Δphz mutant compared to wild-type cultures, indicating that phenazines are crucial for the formation of biofilms if oxygen as an electron acceptor is unavailable. Furthermore, while the model archaeon Haloferax volcanii does not require archaella for surface attachment under static conditions, we demonstrate that an H. volcanii mutant that lacks archaella is impaired in early stages of biofilm formation under shaking conditions. IMPORTANCE Due to the versatility of the mPAD mount, we anticipate that it will aid the analysis of biofilm formation in a broad range of bacteria and archaea. Thereby, it contributes to answering critical biological questions about the regulatory and structural components of biofilm formation and understanding this process in a wide array of environmental, biotechnological, and medical contexts.

Thymidine utilisation pathway is a novel phenotypic switch of Mycoplasma hominis.

Introduction. Mycoplasma hominis is a bacterium belonging to the class Mollicutes. It causes acute and chronic infections of the urogenital tract. The main features of this bacterium are an absence of cell wall and a reduced genome size (517-622 protein-encoding genes). Previously, we have isolated morphologically unknown M. hominis colonies called micro-colonies (MCs) from the serum of patients with inflammatory urogenital tract infection.Hypothesis. MCs are functionally different from the typical colonies (TCs) in terms of metabolism and cell division.Aim. To determine the physiological differences between MCs and TCs of M. hominis and elucidate the pathways of formation and growth of MCs by a comparative proteomic analysis of these two morphological forms.Methodology. LC-MS proteomic analysis of TCs and MCs using an Ultimate 3000 RSLC nanoHPLC system connected to a QExactive Plus mass spectrometer.Results. The study of the proteomic profiles of M. hominis colonies allowed us to reconstruct their energy metabolism pathways. In addition to the already known pentose phosphate and arginine deamination pathways, M. hominis can utilise ribose phosphate and deoxyribose phosphate formed by nucleoside catabolism as energy sources. Comparative proteomic HPLC-MS analysis revealed that the proteomic profiles of TCs and MCs were different. We assume that MC cells preferably utilised deoxyribonucleosides, particularly thymidine, as an energy source rather than arginine or ribonucleosides. Utilisation of deoxyribonucleosides is less efficient as compared with that of ribonucleosides and arginine in terms of energy production. Thymidine phosphorylase DeoA is one of the key enzymes of deoxyribonucleosides utilisation. We obtained a DeoA overexpressing mutant that exhibited a phenotype similar to that of MCs, which confirmed our hypothesis.Conclusion. In addition to the two known pathways for energy production (arginine deamination and the pentose phosphate pathway) M. hominis can use deoxyribonucleosides and ribonucleosides. MC cells demonstrate a reorganisation of energy metabolism: unlike TC cells, they preferably utilise deoxyribonucleosides, particularly thymidine, as an energy source rather than arginine or ribonucleosides. Thus MC cells enter a state of energy starvation, which helps them to survive under stress, and in particular, to be resistant to antibiotics.

Histopathology of a threatened surf clam, toheroa (Paphies ventricosa) from Aotearoa New Zealand.

The toheroa (Paphies ventricosa) is endemic to Aotearoa (New Zealand). Following decades of overfishing in the 1900 s, commercial and recreational fishing of toheroa is now prohibited. For unknown reasons, protective measures in place for over 40 years have not ensured the recovery of toheroa populations. For the first time, a systematic pathology survey was undertaken to provide a baseline of toheroa health in remaining major populations. Using histopathology, parasites and pathologies in a range of tissues are assessed and quantified spatio-temporally. Particular focus is placed on intracellular microcolonies of bacteria (IMCs). Bayesian ordinal logistic regression is used to model IMC infection and several facets of toheroa health. Model outputs show condition to be the most important predictor of IMC intensity in toheroa tissues. The precarious state of many toheroa populations around Aotearoa should warrant greater attention from scientists, conservationists, and regulators. It is hoped that this study will provide some insight into the current health status of a treasured and iconic constituent of several expansive surf beaches in Aotearoa.

Characterisation and distribution of the bacterial genus Endozoicomonas in a threatened surf clam.

The toheroa Paphies ventricosa is a large Aotearoa New Zealand (ANZ) endemic surf clam of cultural importance to many Maori, the Indigenous people of ANZ. Extensive commercial and recreational harvesting in the 20th century dramatically reduced populations, leading to the collapse and closure of the fishery. Despite being protected for >40 yr, toheroa have inexplicably failed to recover. In 2017, intracellular microcolonies (IMCs) of bacteria were detected in 'sick' toheroa in northern ANZ. Numerous mass mortality events (MMEs) have recently been recorded in ANZ shellfish, with many events linked by the presence of IMCs resembling Rickettsia-like organisms (RLOs). While similar IMCs have been implicated in MMEs in surf clams elsewhere, the impact of these IMCs on the health or recovery of toheroa is unknown. A critical first step towards understanding the significance of a pathogen in a host population is pathogen identification and characterisation. To begin this process, we examined 16S rRNA gene sequences of the putative IMCs from 4 toheroa populations that showed 97% homology to Endozoicomonas spp. sequences held in GenBank. Phylogenetic analysis identified closely related Endozoicomonas strains from the North and South Island, ANZ, and in situ hybridization, using 16S rRNA gene probes, confirmed the presence of the sequenced IMC gene in the gill and digestive gland tissues of toheroa. Quantitative PCR revealed site-specific and seasonal abundance patterns of Endozoicomonas spp. in toheroa populations. Although implicated in disease outbreaks elsewhere, the role of Endozoicomonas spp. within the ANZ shellfish mortality landscape remains uncertain.

Confocal Laser Microscopy Analysis of Listeria monocytogenes Biofilms and Spatially Organized Communities.

The behavior of Listeria monocytogenes communities in the food chain is closely associated with their spatial organization. Whether as biofilms on industrial surfaces or as microcolonies in food matrices, the resulting physiological diversification combined with the presence of extracellular polymeric substances (EPS) triggers emergent community functions involved in the pathogen survival and persistence (e.g., tolerance to dehydration, biocides, or preservatives). In this contribution, we present a noninvasive confocal laser microscopy (CLM) protocol allowing exploration of the spatial organization of L. monocytogenes communities on various inert or nutritive materials relevant for the food industry.

Bacteria Modify Candida albicans Hypha Formation, Microcolony Properties, and Survival within Macrophages.

Phagocytic cells are crucial components of the innate immune system preventing Candida albicans mucosal infections. Streptococcus gordonii and Pseudomonas aeruginosa often colonize mucosal sites, along with C. albicans, and yet interkingdom interactions that might alter the survival and escape of fungi from macrophages are not understood. Murine macrophages were coinfected with S. gordonii or P. aeruginosa, along with C. albicans to evaluate changes in fungal survival. S. gordonii increased C. albicans survival and filamentation within macrophage phagosomes, while P. aeruginosa reduced fungal survival and filamentation. Coinfection with S. gordonii resulted in greater escape of C. albicans from macrophages and increased size of fungal microcolonies formed on macrophage monolayers, while coinfection with P. aeruginosa reduced macrophage escape and produced smaller microcolonies. Microcolonies formed in the presence of P. aeruginosa cells outside macrophages also had significantly reduced size that was not found with P. aeruginosa phenazine deletion mutants. S. gordonii cells, as well as S. gordonii heat-fixed culture supernatants, increased C. albicans microcolony biomass but also resulted in microcolony detachment. A heat-resistant, trypsin-sensitive pheromone processed by S. gordonii Eep was needed for these effects. The majority of fungal microcolonies formed on human epithelial monolayers with S. gordonii supernatants developed as large floating structures with no detectable invasion of epithelium, along with reduced gene expression of C. albicansHYR1, EAP1, and HWP2 adhesins. However, a subset of C. albicans microcolonies was smaller and had greater epithelial invasiveness compared to microcolonies grown without S. gordonii Thus, bacteria can alter the killing and escape of C. albicans from macrophages and contribute to changes in C. albicans pathogenicity.IMPORTANCECandida albicans is the predominant fungus colonizing the oral cavity that can have both synergistic and antagonistic interactions with other bacteria. Interkingdom polymicrobial associations modify fungal pathogenicity and are believed to increase microbial resistance to innate immunity. However, it is not known how these interactions alter fungal survival during phagocytic killing. We demonstrated that secreted molecules of S. gordonii and P. aeruginosa alter C. albicans survival within the phagosome of macrophages and alter fungal pathogenic phenotypes, including filamentation and microcolony formation. Moreover, we provide evidence for a dual interaction between S. gordonii and C. albicans such that S. gordonii signaling peptides can promote C. albicans commensalism by decreasing microcolony attachment while increasing invasion in epithelial cells. Our results identify bacterial diffusible factors as an attractive target to modify virulence of C. albicans in polymicrobial infections.

Sho1p Connects Glycolysis to Ras1-cAMP Signaling and Is Required for Microcolony Formation in Candida albicans.

Candida albicans is an opportunistic, dimorphic fungus that causes candidiasis in immunocompromised people. C. albicans forms specialized structures called microcolonies that are important for surface adhesion and virulence. Microcolonies form in response to specific environmental conditions and require glycolytic substrates for optimal growth. However, fungal signaling pathways involved in sensing and transmitting these environmental cues to induce microcolony formation have not been identified. Here, we show that the C. albicans Ras1-cAMP cascade is required for microcolony formation, while the Cek1-MAP kinase pathway is not required, and Hog1 represses microcolony formation. The membrane protein Sho1, known to regulate the Cek1 pathway in yeasts, was indispensable for C. albicans microcolony formation but regulated the Ras1-cAMP pathway instead, based upon diminished intracellular levels of cAMP and reduced expression of core microcolony genes, including HWP1, PGA10, and ECE1, in C. albicanssho1Δ cells. Based upon predicted physical interactions between Sho1 and the glycolytic enzymes Pfk1, Fba1, Pgk1, and Cdc19, we hypothesized that Sho1 regulates Ras1-cAMP by establishing cellular energy levels produced by glycolysis. Indeed, microcolony formation was restored in C. albicanssho1Δ cells by addition of exogenous intermediates of glycolysis, including downstream products of each predicted interacting enzyme (fructose 1,6 bisphosphate, glyceraldehyde phosphate, 3-phosphoglyceric acid, and pyruvate). Thus, C. albicans Sho1 is an upstream regulator of the Ras1-cAMP signaling pathway that connects glycolytic metabolism to the formation of pathogenic microcolonies.IMPORTANCEC. albicans microcolonies form extensive hyphal structures that enhance surface adherence and penetrate underlying tissues to promote fungal infections. This study examined the environmental conditions that promote microcolony formation and how these signals are relayed, in order to disrupt signaling and reduce pathogenesis. We found that a membrane-localized protein, Sho1, is an upstream regulator of glycolysis and required for Ras1-cAMP signaling. Sho1 controlled the Ras1-dependent expression of core microcolony genes involved in adhesion and virulence. This new regulatory function for Sho1 linking glycolysis to microcolony formation reveals a novel role for this fungal membrane protein.

New advances on the Brettanomyces bruxellensis biofilm mode of life.

The wine spoilage yeast Brettanomyces bruxellensis can be found at several steps in the winemaking process due to its resistance to multiple stress conditions. The ability to form biofilm is a potential resistance strategy, although it has been given little attention so far for this yeast. In this work, the capacity to form biofilm and its structure were explored in YPD medium and in wine. Using microsatellite analysis, 65 isolates were discriminated into 5 different genetic groups from which 12 strains were selected. All 12 strains were able to form biofilm in YPD medium on a polystyrene surface. The presence of microcolonies, filamentous cells and extracellular polymeric substances, constituting the structure of the biofilm despite a small thickness, were highlighted using confocal and electronic microscopy. Moreover, different cell morphologies according to genetic groups were highlighted. The capacity to form biofilm in wine was also revealed for two selected strains. The impact of wine on biofilms was demonstrated with firstly considerable biofilm cell release and secondly growth of these released biofilm cells, both in a strain dependent manner. Finally, B. bruxellensis has been newly described as a producer of chlamydospore-like structures in wine, for both planktonic and biofilm lifestyles.

The prevalence and clinical significance of microcolonies when tested according to contemporary interpretive breakpoints for fluconazole against Candida species using E-test.

Changes in the interpretive-breakpoints for antifungals against various Candida species have raised the need to examine the significance of the phenomenon of the growth of microcolonies in agar diffusion inhibition zones, which has generally been considered negligible. The objective was to determine the incidence of cases in which microcolonies demonstrate fluconazole resistance according to current interpretive-breakpoints and whether their growth is associated with therapeutic failure. The fluconazole minimum inhibitory concentrations (MICs) of 100 blood culture isolates of Candida were performed by E-test on Roswell Park Memorial Institute (RPMI) agar and examined for the appearance of microcolonies. Fluconazole MICs of microcolonies were then determined over three generations. The significance of the phenomenon of microcolonies was determined according to clinical data retrieved from electronic files. Microcolonies were a common phenomenon among Candida isolates following incubation on RPMI agar, with a higher frequency among C. albicans isolates as compared to non-albicans Candida across generations (57-93% vs 31-93%, respectively) and a similar fluconazole susceptibility rate over three generations. The rate of microcolonies was similar in both patients with successful and unsuccessful outcome (41% vs 42%, respectively). Microcolonies are a common phenomenon. No increase in MIC was demonstrated throughout three generations of microcolony inoculation on RPMI, and no difference in clinical outcome was observed.

Digital, Rapid, Accurate, and Label-Free Enumeration of Viable Microorganisms Enabled by Custom-Built On-Glass-Slide Culturing Device and Microscopic Scanning.

Accurately measuring the number of viable microorganisms plays an essential role in microbiological studies. Since the conventional agar method of enumerating visible colonies is time-consuming and not accurate, efforts have been made towards overcoming these limitations by counting the invisible micro-colonies. However, none of studies on micro-colony counting was able to save significant time or provide accurate results. Herein, we developed an on-glass-slide cell culture device that enables rapid formation of micro-colonies on a 0.38 mm-thick gel film without suffering from nutrient and oxygen deprivation during bacteria culturing. Employing a phase contrast imaging setup, we achieved rapid microscopic scanning of micro-colonies within a large sample area on the thin film without the need of fluorescent staining. Using Escherichia coli (E. coli) as a demonstration, our technique was able to shorten the culturing time to within 5 h and automatically enumerate the micro-colonies from the phase contrast images. Moreover, this method delivered more accurate counts than the conventional visible colony counting methods. Due to these advantages, this imaging-based micro-colony enumeration technique provides a new platform for the quantification of viable microorganisms.

Bacterial Microcolonies in Gel Beads for High-throughput Screening.

High-throughput screening of a DNA library expressed in a bacterial population for identifying potentially rare members displaying a property of interest is a crucial step for success in many experiments such as directed evolution of proteins and synthetic circuits and deep mutational scanning to identify gain- or loss-of-function mutants. Here, I describe a protocol for high-throughput screening of bacterial (E. coli) microcolonies in gel beads. Single cells are encapsulated into monodisperse water-in-oil emulsion droplets produced with a microfluidic device. The aqueous solution also contains agarose that gelates upon cooling on ice, so that solid gel beads form inside the droplets. During incubation of the emulsion, the cells grow into monoclonal microcolonies inside the beads. After isolation of the gel beads from the emulsion and their sorting by fluorescence activated cell sorting (FACS), the bacteria are recovered from the gel beads and are then ready for a further round of sorting, mutagenesis or analysis. In order to sort by FACS, this protocol requires a fluorescent readout, such as the expression of a fluorescent reporter protein. Measuring the average fluorescent signals of microcolonies reduces the influence of high phenotypic cell-to-cell variability and increases the sensitivity compared to the sorting of single cells. We applied this method to sort a pBAD promoter library at ON and OFF states (Duarte et al., 2017).

A Sub-population of Group A Streptococcus Elicits a Population-wide Production of Bacteriocins to Establish Dominance in the Host.

Bacteria use quorum sensing (QS) to regulate gene expression. We identified a group A Streptococcus (GAS) strain possessing the QS system sil, which produces functional bacteriocins, through a sequential signaling pathway integrating host and bacterial signals. Host cells infected by GAS release asparagine (ASN), which is sensed by the bacteria to alter its gene expression and rate of proliferation. We show that upon ASN sensing, GAS upregulates expression of the QS autoinducer peptide SilCR. Initial SilCR expression activates the autoinduction cycle for further SilCR production. The autoinduction process propagates throughout the GAS population, resulting in bacteriocin production. Subcutaneous co-injection of mice with a bacteriocin-producing strain and the globally disseminated M1T1 GAS clone results in M1T1 killing within soft tissue. Thus, by sensing host signals, a fraction of a bacterial population can trigger an autoinduction mechanism mediated by QS, which acts on the entire bacterial community to outcompete other bacteria within the infection.

Live-cell Imaging of Neisseria meningitidis Microcolony Dispersal Induced by Lactate or Other Molecules.

To efficiently colonize the nasopharyngeal epithelium, the human restricted pathogen Neisseria meningitidis follows a multistep adhesion cascade. First, the bacteria adhere to host cells and aggregate into spherical shaped structures called microcolonies. Several hours later, single bacteria start dispersing from the microcolonies and form a monolayer on top of the host cells. Once in proximity to host cells meningococci can adhere tightly to the epithelial surface or become internalized. This can eventually result in invasion of the mucosal surfaces and gain access to the bloodstream, causing a life-threatening disease. Lactate, a metabolite derived from human epithelial cells, has been previously shown to induce rapid dispersal of N. meningitidis from microcolonies. Here, we describe a host-cell free method based on live-cell imaging to examine the effect of host derived lactate on the timing of N. meningitides microcolony dispersal. Although in this protocol we use lactate, it can be easily modified to test the effects of other molecules.