微生物学

Campylobacter jejuni, a zoonotic foodborne pathogen, is the worldwide leading cause of acute human bacterial gastroenteritis. Biofilms are a significant reservoir for survival and transmission of this pathogen, contributing to its overall antimicrobial resistance. Natural compounds such as essential oils, phytochemicals, polyphenolic extracts, and D-amino acids have been shown to have the potential to control biofilms formed by bacteria, including Campylobacter spp. This work presents a proposed guideline for assessing and characterizing bacterial biofilm formation in the presence of naturally occurring inhibitory molecules using C. jejuni as a model. The following protocols describe: i) biofilm formation inhibition assay, designed to assess the ability of naturally occurring molecules to inhibit the formation of biofilms; ii) biofilm dispersal assay, to assess the ability of naturally occurring inhibitory molecules to eradicate established biofilms; iii) confocal laser scanning microscopy (CLSM), to evaluate bacterial viability in biofilms after treatment with naturally occurring inhibitory molecules and to study the structured appearance (or architecture) of biofilm before and after treatment.

Microbiologists are learning to appreciate the importance of “functional amyloids” that are produced by numerous bacterial species and have impacts beyond the microbial world. These structures are used by bacteria to link together, presumably to increase survival, protect against harsh conditions, and perhaps to influence cell-cell communication. Bacterial functional amyloids are also beginning to be appreciated in the context of host-pathogen interactions, where there is evidence that they can trigger the innate immune system and are recognized as non-self-molecular patterns. The characteristic three-dimensional fold of amyloids renders them similar across the bacterial kingdom and into the eukaryotic world, where amyloid proteins can be undesirable and have pathological consequences. The bacterial protein curli, produced by pathogenic Salmonella enterica and Escherichia coli strains, was one of the first functional amyloids discovered. Curli have since been well characterized in terms of function, and we are just starting to scratch the surface about their potential impact on eukaryotic hosts. In this manuscript, we present step-by-step protocols with pictures showing how to purify these bacterial surface structures. We have described the purification process from S. enterica, acknowledging that the same method can be applied to E. coli. In addition, we describe methods for detection of curli within animal tissues (i.e., GI tract) and discuss purifying curli intermediates in a S. enterica msbB mutant strain as they are more cytotoxic than mature curli fibrils. Some of these methods were first described elsewhere, but we wanted to assemble them together in more detail to make it easier for researchers who want to purify curli for use in biological experiments. Our aim is to provide methods that are useful for specialists and non-specialists as bacterial amyloids become of increasing importance.

Bacterial swarming refers to a rapid spread, with coordinated motion, of flagellated bacteria on a semi-solid surface (Harshey, 2003). There has been extensive study on this particular mode of motility because of its interesting biological and physical relevance, e.g., enhanced antibiotic resistance (Kearns, 2010) and turbulent collective motion (Steager et al., 2008). Commercial equipment for the live recording of swarm expansion can easily cost tens of thousands of dollars (Morales-Soto et al., 2015); yet, often the conditions are not accurately controlled, resulting in poor robustness and a lack of reproducibility. Here, we describe a reliable design and operations protocol to perform reproducible bacterial swarming assays using time-lapse photography. This protocol consists of three main steps: 1) building a “homemade,” environment-controlled photographing incubator; 2) performing a bacterial swarming assay; and 3) calculating the swarming rate from serial photos taken over time. An efficient way of calculating the bacterial swarming rate is crucial in performing swarming phenotype-related studies, e.g., screening swarming-deficient isogenic mutant strains. The incubator is economical, easy to operate, and has a wide range of applications. In fact, this system can be applied to many slowly evolving processes, such as biofilm formation and fungal growth, which need to be monitored by camera under a controlled temperature and ambient humidity.

Oxygenic photogranules (OPGs) are dense, three-dimensional aggregates containing a syntrophic, light-driven microbial community. Their temporal and spatial development interests microbial ecologists working at the bioprocess engineering interface, as this knowledge can be used to optimize biotechnological applications, such as wastewater treatment and biomass valorization. The method presented here enables the high-throughput quantification of photogranulation. OPGs are produced from a loose sludge-like microbial matrix in hydrostatic batch cultures exposed to light. This matrix transforms into a consolidated, roughly spherical aggregate over time. Photogranulation is quantified by time-lapse imaging coupled to automated image analysis. This allows studying the development of many OPGs simultaneously and in a fully automated way to systematically test what factors drive photogranulation. The protocol can also be used to quantify other types of (a)biotic aggregation.

Pseudomonas aeruginosa is a human pathogen capable to form robust biofilms. P. aeruginosa biofilms represent a serious problem because of the adverse effects on human health and industry, from sanitary and economic points of view. Typical strategies to break down biofilms have been long used, such as the use of disinfectants or antibiotics, but also, according to their high resistance to standard antimicrobial approaches, alternative strategies employing photocatalysis or control of biofilm formation by modifying surfaces, have been proposed. Colony forming units (cfu) counting and live/dead staining, two classic techniques used for biofilm quantification, are detailed in this work. Both methods assess cell viability, a key factor to analyze the microbial susceptibility to given treatment, then, they represent a good approach for evaluation of an antibiofilm strategy.

A new direct contact assessment of soil toxicity using sulfur oxidizing bacteria (SOB) is proposed for analyzing the toxicity of soils. The proposed method is based on the ability of SOB to oxidize elemental sulfur to sulfuric acid in the presence of oxygen. Since sulfate ions are produced from sulfur by SOB oxidation activity, changes in electrical conductivity (EC) serve as a proxy to assess toxicity in water. However, in soil medium, EC values are not reliable due to the adsorption of SO₄^2- ions by soils. Here, we suggest a new parameter which measures oxygen consumption by SOB for 6 hours to assess soil toxicity by using a lubricated glass syringe method. The proposed method is rapid, simple, cost- effective as well as sensitive and capable of assessing direct contact soil toxicity.

Candida albicans is the most common cause of fungal infections worldwide. Infection by C. albicans is closely associated with its ability to form a biofilm, closely packed communities of cells attached to the surfaces of human tissues and implanted devices, in or on the host. When tested for susceptibility to antifungals, such as polyenes, azoles, and allylamines, C. albicanscells in a biofilm are more resistant to antifungal agents than C. albicans cells in the planktonic form. Cyclic Adenosine monophosphate (cAMP) is one of the key elements for triggering hyphal and biofilm formation in C. albicans. It is hard to detect or extract molecular markers (e.g., cAMP) from C. albicans biofilms because the biofilms have a complex three-dimensional architecture with an extracellular matrix surrounding the cell walls of the cells in the biofilm. Here, we present an improved protocol that can effectively measure the level of intracellular cAMP in C. albicans biofilms.

Pseudomonas syringae is a model plant pathogen that infects more than 50 plant species worldwide, thus leading to significant yield loss. Pseudomonas biofilm always adheres to the surfaces of medical devices or host cells, thereby contributing to infection. Biofilm formation can be visualized on numerous matrixes, including coverslips, silicone tubes, polypropylene and polystyrene. Confocal laser scanning microscopy can be used to visualize and analyze biofilm structure. In this study, we modified and applied the current method of P. aeruginosa biofilm measurement to P. syringae, and developed a convenient protocol to visualize P. syringae biofilm formation using a borosilicate glass tube as the matrix coupled with crystal violet staining.

Biofilms are bacterial communities in the shape of exopolysaccharide matrix-encased aggregates attached onto interphases able to resist environmental aggressions. The development of bacteria in the shape of biofilms deeply affects the performance of many industrial processes which work with fluidic systems, where bacteria may settle and prosper. As a consequence industrial equipment experiments low performance issues and substantial maintenance costs.

The study of how bacteria of industrial interest such as Pseudomonas putida spread in these fluidic systems is highly dependent on the chosen experimental system to retrieve such data, thus using scaled prototypes becomes an essential step towards the design of a more efficient system to handle biofilms, either to control them or to prevent them. This protocol describes how to assemble, operate and maintain a device to grow and monitor the biofilm spreading pattern of this bacterium (as a function of the fluid hydrodynamics) in a custom-made chamber larger than those typically used in laboratory environments, and how to analyze the information gathered from it in a straightforward fashion. Description of the protocol was thought to be used as a working template not only for the presented case study but for any other potential experiment in different contexts and diverse scales following similar design principles.

Biofilm formation is a well-known bacterial strategy that protects cells from hostile environments. During infection, bacteria found in a biofilm community are less sensitive to antibiotics and to the immune response, often allowing them to colonize and persist in the host niche. Not surprisingly, biofilm formation on medical devices, such as urinary catheters, is a major problem in hospital settings. To be able to eliminate such biofilms, it is important to understand the key bacterial factors that contribute to their formation. A common practice in the lab setting is to study biofilms grown in laboratory media. However, these media do not fully reflect the host environment conditions, potentially masking relevant biological determinants. This is the case during urinary catheterization, where a key element for Enterococcus faecalis and Staphylococcus aureus colonization and biofilm formation is the release of fibrinogen (Fg) into the bladder and its deposition on the urinary catheter. To recapitulate bladder conditions during catheter-associated urinary tract infection (CAUTI), we have developed a fibrinogen-coated catheter and 96-well plate biofilm assay in urine. Notably, enterococcal biofilm factors identified in these in vitro assays proved to be important for biofilm formation in vivo in a mouse model of CAUTI. Thus, the method described herein can be used to uncover biofilm-promoting factors that are uniquely relevant in the host environment, and that can be exploited to develop new antibacterial therapies.