Accelerating the Detection of Bacteria in Food Using Artificial Intelligence and Optical Imaging.

In assessing food microbial safety, the presence of Escherichia coli is a critical indicator of fecal contamination. However, conventional detection methods require the isolation of bacterial macrocolonies for biochemical or genetic characterization, which takes a few days and is labor-intensive. In this study, we show that the real-time object detection and classification algorithm You Only Look Once version 4 (YOLOv4) can accurately identify the presence of E. coli at the microcolony stage after a 3-h cultivation. Integrating with phase-contrast microscopic imaging, YOLOv4 discriminated E. coli from seven other common foodborne bacterial species with an average precision of 94%. This approach also enabled the rapid quantification of E. coli concentrations over 3 orders of magnitude with an R2 of 0.995. For romaine lettuce spiked with E. coli (10 to 103 CFU/g), the trained YOLOv4 detector had a false-negative rate of less than 10%. This approach accelerates analysis and avoids manual result determination, which has the potential to be applied as a rapid and user-friendly bacterial sensing approach in food industries. IMPORTANCE A simple, cost-effective, and rapid method is desired to identify potential pathogen contamination in food products and thus prevent foodborne illnesses and outbreaks. This study combined artificial intelligence (AI) and optical imaging to detect bacteria at the microcolony stage within 3 h of inoculation. This approach eliminates the need for time-consuming culture-based colony isolation and resource-intensive molecular approaches for bacterial identification. The approach developed in this study is broadly applicable for the identification of diverse bacterial species. In addition, this approach can be implemented in resource-limited areas, as it does not require expensive instruments and significantly trained human resources. This AI-assisted detection not only achieves high accuracy in bacterial classification but also provides the potential for automated bacterial detection, reducing labor workloads in food industries, environmental monitoring, and clinical settings.

Lactate-Induced Dispersal of Neisseria meningitidis Microcolonies Is Mediated by Changes in Cell Density and Pilus Retraction and Is Influenced by Temperature Change.

Neisseria meningitidis is the etiologic agent of meningococcal meningitis and sepsis. Initial colonization of meningococci in the upper respiratory tract epithelium is crucial for disease development. The colonization occurs in several steps and expression of type IV pili (Tfp) is essential for both attachment and microcolony formation of encapsulated bacteria. Previously, we have shown that host-derived lactate induces synchronized dispersal of meningococcal microcolonies. In this study, we demonstrated that lactate-induced dispersal is dependent on bacterial concentration but not on the quorum-sensing system autoinducer-2 or the two-component systems NarP/NarQ, PilR/PilS, NtrY/NtrX, and MisR/MisS. Further, there were no changes in expression of genes related to assembly, elongation, retraction, and modification of Tfp throughout the time course of lactate induction. By using pilT and pptB mutants, however, we found that lactate-induced dispersal was dependent on PilT retraction but not on phosphoglycerol modification of Tfp even though the PptB activity was important for preventing reaggregation postdispersal. Furthermore, protein synthesis was required for lactate-induced dispersal. Finally, we found that at a lower temperature, lactate-induced dispersal was delayed and unsynchronized, and bacteria reformed microcolonies. We conclude that lactate-induced microcolony dispersal is dependent on bacterial concentration, PilT-dependent Tfp retraction, and protein synthesis and is influenced by environmental temperature.

Multiple holins contribute to extracellular DNA release in Pseudomonas aeruginosa biofilms.

Bacterial biofilms are composed of aggregates of cells encased within a matrix of extracellular polymeric substances (EPS). One key EPS component is extracellular DNA (eDNA), which acts as a 'glue', facilitating cell-cell and cell-substratum interactions. We have previously demonstrated that eDNA is produced in Pseudomonas aeruginosa biofilms via explosive cell lysis. This phenomenon involves a subset of the bacterial population explosively lysing, due to peptidoglycan degradation by the endolysin Lys. Here we demonstrate that in P. aeruginosa three holins, AlpB, CidA and Hol, are involved in Lys-mediated eDNA release within both submerged (hydrated) and interstitial (actively expanding) biofilms, albeit to different extents, depending upon the type of biofilm and the stage of biofilm development. We also demonstrate that eDNA release events determine the sites at which cells begin to cluster to initiate microcolony formation during the early stages of submerged biofilm development. Furthermore, our results show that sustained release of eDNA is required for cell cluster consolidation and subsequent microcolony development in submerged biofilms. Overall, this study adds to our understanding of how eDNA release is controlled temporally and spatially within P. aeruginosa biofilms.

Role of the Gonococcal Neisserial Heparin Binding Antigen in Microcolony Formation, and Serum Resistance and Adherence to Epithelial Cells.

The sexually transmitted infection gonorrhoea is on the rise worldwide and an increased understanding of the mechanisms of colonization and pathogenesis of Neisseria gonorrhoeae is required to aid development of new treatment and prevention strategies. In the current study, we investigate the neisserial heparin-binding antigen (NHBA) of N. gonorrhoeae and confirm its role in binding to several glycans, including heparin, and identify interactions of NHBA with both gonococcal and host cells. Furthermore, we report that a gonococcal nhba mutant displays decreased cell aggregation and microcolony formation, as well as reduced survival in human serum and reduced adherence to human cervical and urethral epithelial cells, relative to the wild-type strain. These data indicate that the gonococcal NHBA contributes to several aspects of the colonization and survival of N. gonorrhoeae and may be a target for new antimicrobial or vaccines.

Food Microstructure and Fat Content Affect Growth Morphology, Growth Kinetics, and Preferred Phase for Cell Growth of Listeria monocytogenes in Fish-Based Model Systems.

Food microstructure significantly affects microbial growth dynamics, but knowledge concerning the exact influencing mechanisms at a microscopic scale is limited. The food microstructural influence on Listeria monocytogenes (green fluorescent protein strain) growth at 10°C in fish-based food model systems was investigated by confocal laser scanning microscopy. The model systems had different microstructures, i.e., liquid, xanthan (high-viscosity liquid), aqueous gel, and emulsion and gelled emulsion systems varying in fat content. Bacteria grew as single cells, small aggregates, and microcolonies of different sizes (based on colony radii [size I, 1.5 to 5.0 µm; size II, 5.0 to 10.0 µm; size III, 10.0 to 15.0 µm; and size IV, ≥15 µm]). In the liquid, small aggregates and size I microcolonies were predominantly present, while size II and III microcolonies were predominant in the xanthan and aqueous gel. Cells in the emulsions and gelled emulsions grew in the aqueous phase and on the fat-water interface. A microbial adhesion to solvent assay demonstrated limited bacterial nonpolar solvent affinities, implying that this behavior was probably not caused by cell surface hydrophobicity. In systems containing 1 and 5% fat, the largest cell volume was mainly represented by size I and II microcolonies, while at 10 and 20% fat a few size IV microcolonies comprised nearly the total cell volume. Microscopic results (concerning, e.g., growth morphology, microcolony size, intercolony distances, and the preferred phase for growth) were related to previously obtained macroscopic growth dynamics in the model systems for an L. monocytogenes strain cocktail, leading to more substantiated explanations for the influence of food microstructural aspects on lag phase duration and growth rate.IMPORTANCEListeria monocytogenes is one of the most hazardous foodborne pathogens due to the high fatality rate of the disease (i.e., listeriosis). In this study, the growth behavior of L. monocytogenes was investigated at a microscopic scale in food model systems that mimic processed fish products (e.g., fish paté and fish soup), and the results were related to macroscopic growth parameters. Many studies have previously focused on the food microstructural influence on microbial growth. The novelty of this work lies in (i) the microscopic investigation of products with a complex composition and/or structure using confocal laser scanning microscopy and (ii) the direct link to the macroscopic level. Growth behavior (i.e., concerning bacterial growth morphology and preferred phase for growth) was more complex than assumed in common macroscopic studies. Consequently, the effectiveness of industrial antimicrobial food preservation technologies (e.g., thermal processing) might be overestimated for certain products, which may have critical food safety implications.

Thin polyester filters as versatile sample substrates for high-pressure freezing of bacterial biofilms, suspended microorganisms and adherent eukaryotic cells.

High-pressure freezing limits the size of biological samples, because only small samples can be frozen without ice damage. Additionally, these samples must fit into the dimensions of the sample holder provided by the high-pressure freezer. We explored the potential of a 10 µm thin polyester filter membrane (PE-filter) as a versatile sample substrate for high-pressure freezing. Planktonic bacteria, bacterial spores and suspended eukaryotic cells could be concentrated on the PE-filter, whereas biofilm, bacterial microcolonies and HeLa cells were able to grow directly on the PE-filter. These microorganism-loaded PE-filters were used for high-pressure freezing, freeze-substitution and plastic embedding in Epon or Lowicryl. Embedded filters were cross-sectioned so that the interface between microorganism and substrate as well as the overlying medium was revealed. Although the structural preservation was good for thin samples and samples with lower water content, such as biofilms, adherent HeLa-cell cultures were likewise sufficiently preserved for transmission electron microscopy imaging. The fact that microorganism-loaded PE-filters could be also examined with confocal laser scanning fluorescence microscopy under fully hydrated conditions, and freeze-substituted PE-filters samples with scanning electron microscopy, demonstrates the versatility of the PE-filter as a sample substrate for a wide array of microorganisms. LAY DESCRIPTION: In order to investigate biological samples in the transmission electron microscope it is imperative to remove all their water content, or the specimens will be destroyed by boiling in the high vacuum of the microscope. In order to avoid dramatic morphology-changes due to drying artefacts or the impact of chemical stabilisers, high-pressure freezing (HPF) was developed. This protocol allows freezing biological samples in an instant (within a few milliseconds) down to -196°C while applying high pressure at the same time so that the specimen retains all its water in a solidified noncrystalline form. However, the formation of morphology-destroying ice crystals is only avoided, if the cooling of the sample is faster than the ice crystal formation, which is only possible with very thin samples (up to a maximum of 200 µm in optimal cases). High-pressure freezing is regarded as the gold-standard for sample preparation of cells, tissues and small organisms. However, all of these samples must fit into the dimensions of the specific sample holder of the high-pressure freezer and their transfer into the high-pressure freezing machine must be achieved without significant impact on sample physiology. Additionally, it may also necessary to concentrate and immobilise a biological specimen before they can be placed in the HPF sample holder. Although a few number of strategies and sample substrates have been used for different types of biological samples, we explored the potential of a 10 µm thin polyester filter membrane (PE-filter) as a versatile sample substrate for HPF. In culture medium suspended bacteria, suspended bacterial spores and in medium suspended higher cells could be concentrated on the PE-filter, whereas bacterial biofilm or bacterial microcolonies from an agar plate, and surface-adhering higher cells were able to grow directly on the PE-filter. These microorganism-loaded PE-filters could be directly used for high-pressure freezing, and were finally embedded in a plastic resin like Epon or Lowicryl. Embedded filters were cross-sectioned so that the interface between microorganism and substrate or overlying medium was revealed. Although the structural preservation was good for thin samples and samples with lower water content, such as biofilms, adherent HeLa-cell cultures were likewise sufficiently preserved for transmission electron microscopy imaging. The fact that microorganism-loaded PE-filters could be also examined with confocal laser scanning fluorescence microscopy under fully hydrated conditions, and freeze-substituted PE-filters samples with scanning-electron microscopy, demonstrates the versatility of the PE-filter as a sample substrate for a wide array of microorganisms.

Growth Rate of Bumblebee Larvae is Related to Pollen Amino Acids.

The use of Bombus terrestris L. commercial colonies for outdoor and greenhouse crop pollination is currently widespread. Colony breeding includes bumblebee feeding, mostly by using the honeybee pollen loads of diverse palynological composition. Because the chemical content of pollen is highly variable, the choice of commercial blend should not be random but has to be carefully selected to ensure the optimal development of workers and then pollination efficacy. In this work, we compared the impact of three common commercial blends on the development of bumblebee microcolonies, namely, Actinidia deliciosa L., Cistus sp., and Salix sp. We focus on amino acids (i.e., composition and amount), as they are currently used as an indicator of diet performance. Five parameters were used to determine microcolonies growth rate: 1) number of eggs, 2) number of alive larvae, 3) number of ejected larvae, 4) number of pupae, and 5) total number of offspring. Syrup collection was also monitored to estimate energetic requirement for colony growth. Results revealed that the three commercial blends chemically differed in their amino acid contents, with those displaying higher concentrations (i.e., Salix sp. and A. deliciosa) accelerating microcolony development along with an increase of syrup collection. The advantages of rearing bumblebee commercial colonies using a pollen diet with an optimal amino acid content are discussed.

Consumption of pollen contaminated with field-realistic concentrations of fungicide causes sublethal effects in Bombus impatiens (Hymenoptera: Apidae) microcolonies.

Bumble bees are declining across the globe. The causes of this decline have been attributed to a variety of stressors, including pesticides. Fungicides are a type of pesticide that has been understudied in the context of bumble bee health. As a result, fungicides are often applied to flowering plants without consideration of pollinator exposure. Recent work demonstrates that fungicides have sublethal effects in bumble bees, but little is known about how much fungicide it takes to cause these sublethal effects. To address this gap in the literature, we fed microcolonies of the common eastern bumble bee (Bombus impatiens CressonHymenoptera: ApidaeHymenoptera: ApidaeHymenoptera: ApidaeHymenoptera: Apidae) pollen contaminated with a range of fungicide concentrations. We chose these concentrations based on the range of fungicide concentrations in pollen and nectar that were reported in the literature. Results revealed that later-stage pupae and newly emerged males are potentially sensitive to fungicide exposure, showing smaller size and reduced fat reserves at intermediate levels of contamination. Compared to the control, intermediated levels of fungicide-contaminated pollen led to increased pupal mortality and delayed male emergence. Contrary to expectations, higher fungicide levels did not exhibit a linear relationship with negative impacts, suggesting nuanced effects. Because body size and emergence timing are important aspects of bumble bee reproductive behavior, results have implications for mating success, potentially disrupting colony development.

Type 1 fimbriae-mediated collective protection against type 6 secretion system attacks.

Bacterial competition may rely on secretion systems such as the type 6 secretion system (T6SS), which punctures and releases toxic molecules into neighboring cells. To subsist, bacterial targets must counteract the threats posed by T6SS-positive competitors. In this study, we used a comprehensive genome-wide high-throughput screening approach to investigate the dynamics of interbacterial competition. Our primary goal was to identify deletion mutants within the well-characterized E. coli K-12 single-gene deletion library, the Keio collection, that demonstrated resistance to T6SS-mediated killing by the enteropathogenic bacterium Cronobacter malonaticus. We identified 49 potential mutants conferring resistance to T6SS and focused our interest on a deletion mutant (∆fimE) exhibiting enhanced expression of type 1 fimbriae. We demonstrated that the presence of type 1 fimbriae leads to the formation of microcolonies and thus protects against T6SS-mediated assaults. Collectively, our study demonstrated that adhesive structures such as type 1 fimbriae confer collective protective behavior against T6SS attacks.IMPORTANCEType 6 secretion systems (T6SS) are molecular weapons employed by gram-negative bacteria to eliminate neighboring microbes. T6SS plays a pivotal role as a virulence factor, enabling pathogenic gram-negative bacteria to compete with the established communities to colonize hosts and induce infections. Gaining a deeper understanding of bacterial interactions will allow the development of strategies to control the action of systems such as the T6SS that can manipulate bacterial communities. In this context, we demonstrate that bacteria targeted by T6SS attacks from the enteric pathogen Cronobacter malonaticus, which poses a significant threat to infants, can develop a collective protective mechanism centered on the production of type I fimbriae. These adhesive structures promote the aggregation of bacterial preys and the formation of microcolonies, which protect the cells from T6SS attacks.

Nitric oxide signaling through three receptors regulates virulence, biofilm formation, and phenotypic heterogeneity of Legionella pneumophila.

The causative agent of Legionnaires' disease, Legionella pneumophila, is an environmental bacterium, that replicates in macrophages, parasitizes amoeba, and forms biofilms. L. pneumophila employs the Legionella quorum sensing (Lqs) system and the transcription factor LvbR to control various bacterial traits, including virulence and biofilm architecture. LvbR negatively regulates the nitric oxide (NO) receptor Hnox1, linking quorum sensing to NO signaling. Here, we assessed the response of L. pneumophila to NO and investigated bacterial receptors underlying this process. Chemical NO donors, such as dipropylenetriamine (DPTA) NONOate and sodium nitroprusside (SNP), delayed and reduced the expression of the promoters for flagellin (PflaA) and the 6S small regulatory RNA (P6SRNA). Marker-less L. pneumophila mutant strains lacking individual (Hnox1, Hnox2, or NosP) or all three NO receptors (triple knockout, TKO) grew like the parental strain in media. However, in the TKO strain, the reduction of PflaA expression by DPTA NONOate was less pronounced, suggesting that the NO receptors are implicated in NO signaling. In the ΔnosP mutant, the lvbR promoter was upregulated, indicating that NosP negatively regulates LvbR. The single and triple NO receptor mutant strains were impaired for growth in phagocytes, and phenotypic heterogeneity of non-growing/growing bacteria in amoebae was regulated by the NO receptors. The single NO receptor and TKO mutant strains showed altered biofilm architecture and lack of response of biofilms to NO. In summary, we provide evidence that L. pneumophila regulates virulence, intracellular phenotypic heterogeneity, and biofilm formation through NO and three functionally non-redundant NO receptors, Hnox1, Hnox2, and NosP. IMPORTANCE: The highly reactive diatomic gas molecule nitric oxide (NO) is produced by eukaryotes and bacteria to promote short-range and transient signaling within and between neighboring cells. Despite its importance as an inter-kingdom and intra-bacterial signaling molecule, the bacterial response and the underlying components of the signaling pathways are poorly characterized. The environmental bacterium Legionella pneumophila forms biofilms and replicates in protozoan and mammalian phagocytes. L. pneumophila harbors three putative NO receptors, one of which crosstalks with the Legionella quorum sensing (Lqs)-LvbR network to regulate various bacterial traits, including virulence and biofilm architecture. In this study, we used pharmacological, genetic, and cell biological approaches to assess the response of L. pneumophila to NO and to demonstrate that the putative NO receptors are implicated in NO detection, bacterial replication in phagocytes, intracellular phenotypic heterogeneity, and biofilm formation.

Dynamic single cell analysis in a proximal-tubule-on-chip reveals heterogeneous epithelial colonization strategies of uropathogenic Escherichia coli under shear stress.

The urinary tract is a hydrodynamically challenging microenvironment and uropathogenic Escherichia coli (UPEC) must overcome several physiological challenges in order to adhere and establish a urinary tract infection. Our previous work in vivo revealed a synergy between different UPEC adhesion organelles, which facilitated effective colonization of the renal proximal tubule. To allow high-resolution real-time analysis of this colonization behavior, we established a biomimetic proximal-tubule-on-chip (PToC). The PToC allowed for single-cell resolution analysis of the first stages of bacterial interaction with host epithelial cells, under physiological flow. Time-lapse microscopy and single-cell trajectory analysis in the PToC revealed that while the majority of UPEC moved directly through the system, a minority population initiated heterogeneous adhesion, identified as either rolling or bound. Adhesion was predominantly transient and mediated by P pili at the earliest time-points. These bound bacteria initiated a founder population which rapidly divided, leading to 3D microcolonies. Within the first hours, the microcolonies did not express extracellular curli matrix, but rather were dependent on Type 1 fimbriae as the key element in the microcolony structure. Collectively, our results show the application of Organ-on-chip technology to address bacterial adhesion behaviors, demonstrating a well-orchestrated interplay and redundancy between adhesion organelles that enables UPEC to form microcolonies and persist under physiological shear stress.

Microliter spotting and micro-colony observation: A rapid and simple approach for counting bacterial colony forming units.

For enumerating viable bacteria, traditional dilution plating to count colony forming units (CFUs) has always been the preferred method in microbiology owing to its simplicity, albeit being laborious and time-consuming. Similar CFU counts can be obtained by quantifying growing micro-colonies in conjunction with the benefits of a microscope. Here, we employed a simple method of five to ten microliter spotting of a diluted bacterial culture multiple times on a single Petri dish followed by determining CFU by counting micro-colonies using a phase-contrast microscope. In this method, the CFU of an Escherichia coli culture can be estimated within a four-hour period after spotting. Further, within a ten-hour period after spotting, CFU in a culture of Ralstonia solanacearum, a bacterium with a generation time of around 2 h, can be estimated. The CFU number determined by micro-colonies observed for 106-fold dilutions or lower is similar to that obtained by the dilution plating method for 107-fold dilutions or lower. Micro-colony numbers observed in the early hours of growth (2 h in case of E. coli and 8 h in case of R. solanacearum) were found to remain consistent at later hours (4 h in case of E. coli and 10 h in case of R. solanacearum), where the visibility of the colonies was better due to a noticeable increase in the size of the colonies. This suggested that micro-colonies observed in the early hours indeed represent the bacterial number in the culture. Practical applications to this counting method were employed in studying the rifampicin-resistant mutation rate as well as performing a fluctuation test in E. coli. The spotting method described here to enumerate bacterial CFU results in reduction of labour, time and resources.

A Comparison of Pollen and Syrup Exposure Routes in Bombus impatiens (Hymenoptera: Apidae) Microcolonies: Implications for Pesticide Risk Assessment.

Bumble bees are important pollinators for both native plants and managed agricultural systems. Accumulating evidence has shown that pesticides, including neonicotinoids, can have a range of adverse effects on bumble bee health. Most laboratory studies that assess the effects of chronic neonicotinoid exposure on bumble bees use syrup as the delivery vehicle, rather than pollen. However, in the field, it is likely that bumble bees are exposed to neonicotinoids in both nectar (syrup) and pollen. To examine the potential for different effects based on the vehicle, we compared two studies of chronic exposure to the neonicotinoid acetamiprid in Bombus impatiens microcolonies. We examined correlations between microcolony endpoints and identified associations between the timing of colony pollen and syrup consumption and drone production. Furthermore, in line with previous results, we found that average drone weight was affected at a range of doses only when microcolonies were exposed to acetamiprid via pollen. In general, our analyses point to the importance of the treatment vehicle and suggest that critical effects on developing brood could be missed when neonicotinoid exposure occurs only through syrup.

Protocol for Initiating and Monitoring Bumble Bee Microcolonies with Bombus impatiens (Hymenoptera: Apidae).

Populations of some bumble bee species are in decline, prompting the need to better understand bumble bee biology and for assessing the effects of environmental stressors on these important pollinators. Microcolonies have been successfully used for investigating a range of endpoints, including behavior, gut microbiome, nutrition, development, pathogens, and the effects of pesticide exposure on bumble bee health. Here, we present a step-by-step protocol for initiating, maintaining, and monitoring microcolonies with Bombus impatiens . This protocol has been successfully used in two pesticide exposure-effects studies and can be easily expanded to investigate other aspects of bumble bee biology.

Poison or Potion: Effects of Sunflower Phenolamides on Bumble Bees and Their Gut Parasite.

Specific floral resources may help bees to face environmental challenges such as parasite infection, as recently shown for sunflower pollen. Whereas this pollen diet is known to be unsuitable for the larval development of bumble bees, it has been shown to reduce the load of a trypanosomatid parasite (Crithidia bombi) in the bumble bee gut. Recent studies suggested it could be due to phenolamides, a group of compounds commonly found in flowering plants. We, therefore, decided to assess separately the impacts of sunflower pollen and its phenolamides on a bumble bee and its gut parasite. We fed Crithidia-infected and -uninfected microcolonies of Bombus terrestris either with a diet of willow pollen (control), a diet of sunflower pollen (natural diet) or a diet of willow pollen supplemented with sunflower phenolamides (supplemented diet). We measured several parameters at both microcolony (i.e., food collection, parasite load, brood development and stress responses) and individual (i.e., fat body content and phenotypic variation) levels. As expected, the natural diet had detrimental effects on bumble bees but surprisingly, we did not observe any reduction in parasite load, probably because of bee species-specific outcomes. The supplemented diet also induced detrimental effects but by contrast to our a priori hypothesis, it led to an increase in parasite load in infected microcolonies. We hypothesised that it could be due to physiological distress or gut microbiota alteration induced by phenolamide bioactivities. We further challenged the definition of medicinal effects and questioned the way to assess them in controlled conditions, underlining the necessity to clearly define the experimental framework in this research field.

Cost-Effective and Versatile Analysis of Archaeal Surface Adhesion Under Shaking and Standing Conditions.

Biofilms are aggregates of cells surrounded by an extracellular matrix providing protection from external stresses. While biofilms are commonly studied in bacteria, archaea also form such cell aggregates both in liquid cultures and on solid surfaces. Biofilm architectures vary when in liquid cultures versus on surfaces as well as when incubated under static conditions versus under shear forces of flowing liquid. Moreover, biofilms develop dynamically over time. Here, we describe surface adhesion assays employing a cost-effective, 3D-printed coverslip holder that can be used under a broad range of standing and shaking culture conditions. This multi-panel adhesion (mPAD) mount further allows the same culture to be sampled at multiple time points, ensuring consistency and comparability between samples and enabling analysis of the dynamics of biofilm formation. Additionally, a traditional surface adhesion assay in a 12-well plate under standing conditions is outlined as well. We anticipate the combination of these protocols to be useful for analyzing a wide array of biofilms and answering a multitude of biological questions.

Single-Cell Analysis of the Antimicrobial and Bactericidal Activities of the Antimicrobial Peptide Magainin 2.

Antimicrobial peptides (AMPs) inhibit the proliferation of or kill bacterial cells. To measure these activities, several methods have been used, which provide only the average value of many cells. Here, we report the development of a method to examine the antimicrobial and bactericidal activities of AMPs at the single-cell level (i.e., single-cell analysis) and apply this strategy to examine the interaction of an AMP, magainin 2 (Mag), with Escherichia coli cells. Using this method, we monitored the proliferation of single cells on agar in a microchamber and measured the distribution of the number of cells in each microcolony using optical microscopy. For method A, we incubated cells in the presence of various concentrations of AMPs for 3 h. The fraction of microcolonies containing only a single cell, Psingle, increased with the Mag concentration and reached 1 at a specific concentration, which corresponded to the MIC. For method B, after the interaction of a cell suspension with an AMP for a specific time, an aliquot was diluted to stop the interaction, and the proliferation of single cells then was monitored after a 3-h incubation; this method permits the definition of Psingle(t), the fraction of dead cells after the interaction. For the interaction of Mag with E. coli cells, Psingle(t) increased with the interaction time, reaching ~1 at 10 and 20 min for 25 and 13 µM Mag, respectively. Thus, these results indicate that a short interaction time between Mag and E. coli cells is sufficient to induce bacterial cell death. IMPORTANCE To elucidate the activity of antimicrobial peptides (AMPs) against bacterial cells, it is important to estimate the interaction time that is sufficient to induce cell death. We have developed a method to examine the antimicrobial and bactericidal activities of AMPs at the single-cell level (i.e., single-cell analysis). Using this method, we monitored the proliferation of single cells on agar in a microchamber and measured the distribution of the number of cells in each microcolony using optical microscopy. We found that during the interaction of magainin 2 (Mag) with E. coli cells, the fraction of dead cells, Psingle(t), increased with the interaction time, rapidly reaching 1 (e.g., 10 min for 25 µM Mag). This result indicates that Mag induces cell death after a short time of interaction.

Sulfoxaflor and nutritional deficiency synergistically reduce survival and fecundity in bumblebees.

A range of anthropogenic factors are causing unprecedented bee declines. Among these drivers the usage of pesticides is believed to be crucial. While the use of key bee-harming insecticides, such as the neonicotinoids, has been reduced by regulatory authorities, novel, less studied substances have occupied their market niche. Understanding the threat of these chemicals to bees is, therefore, crucial to their conservation. Here we focus on sulfoxaflor, a novel insecticide, targeting the same neural receptor as the neonicotinoids. In stark contrast to the growing concerns around its negative impacts on bee health, a recent assessment has resulted in the extension of its authorisations across the USA. However, such assessments may underestimate risks by overlooking interactive impacts of multiple stressors. Here we investigated co-occurring, lethal and sublethal risks of sulfoxaflor and a dietary stress for bumblebees (Bombus terrestris), a key pollinator. Specifically, we employed a novel microcolony design, where, for the first time in bees, pesticide exposure mimicked natural degradation. We orally exposed workers to sulfoxaflor and a sugar-deficient diet in a fully factorial design. Field realistic, worst-case sulfoxaflor exposure caused a sharp increase in bee mortality. At sublethal concentrations, sulfoxaflor negatively affected bee fecundity, but not survival. Nutritional stress reduced bee fecundity and synergistically or additively aggravated impacts of sulfoxaflor on bee survival, egg laying and larval production. Our data show that non-mitigated label uses of sulfoxaflor may have major, yet severely neglected effects on bumblebee health, which may be exacerbated by nutritional stress. By unravelling mechanistic interactions of synergistic risks, our study highlights the need to overcome inherent limitations of Environmental Risk Assessment schemes, which, being based on a "single stressor paradigm", may fail to inform policymakers of the real risks of pesticide use.

Short-term lab assessments and microcolonies are insufficient for the risk assessment of insecticides for bees.

Risk assessment studies addressing effects of agrochemicals on bumblebees frequently use microcolonies. These are queenless colonies consisting of workers only in which typically one worker will lay unfertilized male-destined eggs. In the first tier of risk assessment for bees, short-term laboratory experiments (e.g. microcolonies) are used, the results of which will determine whether higher tier (semi-)field experiments are needed. To evaluate the suitability of microcolonies for risk assessment, a direct comparison between different assessment methods for the neonicotinoid pesticides acetamiprid and thiacloprid was made: microcolonies and queenright colonies under short-term laboratory conditions, queenright colonies under long-term laboratory conditions, and queenright colonies under field conditions. Here, we demonstrate that results from microcolonies contradict results from queenright colonies. While thiacloprid negatively impacted gyne production in queenright colonies, it had a positive effect on microcolony size. By contrast, thiacloprid had no significant effect on fitness parameters of queenright colonies under short-term laboratory conditions when mostly workers are produced. These results thus highlight both the need for long term assessments, allowing evaluation of gyne production, and the risk of reaching erroneous conclusions when using microcolonies. The negative effect of thiacloprid on colony fitness was confirmed under field conditions, where thiacloprid affected the production of reproductives, colony weight gain, worker weight, and foraging behaviour. For acetamiprid, a negative trend on colony fitness could only be shown in a field setup. Therefore, field-realistic setups, which allow colonies to forage freely, are most appropriate to assess sublethal effects of pesticides affecting behaviour and learning.

The Utility of a Bumble Bee (Bombus spp. [Hymenoptera: Apidae]) Brood Test for Evaluating the Effects of Pesticides.

Risk assessment for chemicals in the United States relies upon the honey bee (Apis meliffera L. [Hymenoptera: Apidae]) as a surrogate for other bee species. There is uncertainty in extrapolating honey bee toxicity data to bumble bees due to differences in life history strategies, food consumption, and nest structure. Here we evaluated the design of a queenless bumble bee microcolony test that could be considered for generating larval toxicity data. Three microcolony studies were conducted with Bombus impatiens to evaluate the effects of exposure to 1) diflubenzuron in pollen, 2) dimethoate in pollen, and 3) dimethoate in sucrose. Immature drone bee emergence, worker survival, pollen, and sucrose utilization were measured throughout the study duration. For dimethoate, a 10-d chronic adult bumble bee study was also conducted to compare microcolony endpoints to toxicity endpoints on individual adults. Microcolonies exposed to 10 mg diflubenzuron/kg pollen produced fewer adult drones despite no effects on worker survival. Microcolonies treated with dimethoate at ≥3 mg a.i./kg pollen and ≥0.1 mg a.i./kg sucrose solution produced fewer drones. Exposure to dimethoate in the 10-d chronic adult study resulted in direct mortality to the adult workers at ≥0.1 mg a.i./kg diet. Results from the 10-d study suggest direct effects of dimethoate on workers in the microcolony will alter provisioning of diet to the brood, resulting in lower drone production in the microcolony. Our data suggest that the microcolony study is only appropriate to assess brood effects to bumble bees for substances with low toxicity to adults, as demonstrated with diflubenzuron.