Measuring the Migration and Biofilm Formation of Various Bacteria
Summary
Here, we present a practical method for the isolation and identification of microorganisms within the host. In this way, the physicochemical properties of microorganisms and possible ways of living in the host are clearly described.
Abstract
As microbes that thrive in the host body primarily have adaptive abilities that facilitate their survival, methods for classifying and identifying their nature would be beneficial in facilitating their characterization. Currently, most studies focus only on one specific characterization method; however, the isolation and identification of microorganisms from the host is a continuous process and usually requires several combinatorial characterization methods. Herein, we describe methods of identifying the microbial biofilm-forming ability, the microbial respiration state, and their chemotaxis behavior. The methods are used to identify five microbes, three of which were isolated from the bone tissue of Sprague-Dawley (SD) rats (Corynebacterium stationis, Staphylococcus cohnii subsp. urealyticus, and Enterococcus faecalis) and two from the American Type Culture Collection (ATCC)-Staphylococcus aureus ATCC 25923 and Enterococcus faecalis V583. The microbes isolated from the SD rat bone tissue include the gram-positive microbes. These microbes have adapted to thrive under stressful and nutrient-limiting environments within the bone matrix. This article aims to provide the readers with the specific know-how of determining the nature and behavior of the isolated microbes within a laboratory setting.
Introduction
The mammalian host represented by the human body contains a large number of microorganisms. These microorganisms are widely distributed in the mouth, digestive tract, intestine, and blood of the host and have different effects on the host's health. The oral cavity is host to a plethora of microbes that can modulate the host's susceptibility to infections. Microbes such as Streptococci (e.g., S. mitis/oralis, S. pseudopneumoniae, and S. infantis) and Prevotella spp. colonize the oral cavity, forming a multispecies biofilm on the tongue surface causing bad breath and functioning as a microbial reservoir for microbial infection. These pathogens can infect the jawbone by infiltrating the periodontal ligaments that hold the tooth root in the jawbone1. The characterization of these microbes isolated from the host body is often a tedious process, particularly when the microbes exhibit individual traits requiring specific treatment and growth conditions. Microbes, such as the pathogenic Helicobacter pylori, Clostridium difficile, and Fusobacterium nucleatum, thrive in the gut's harsh environment, with specific oxygen, nutrient, and growth requirements, presenting a challenge in the characterization processes, particularly in investigating the pathogenicity of these microbes. Therefore, a standardized method of isolating and investigating these microorganisms is needed for scientists and medical practitioners to develop new medical treatments.
This protocol uses microbes that thrive in the bone matrix of rats. Traditionally, with the exception of the osseous system represented by the jaw, where the presence of teeth makes the bone matrix more susceptible to infection than other bones1, it is generally believed that the host's healthy bone is a sterile environment. However, studies have found that microorganisms enter the systemic circulation through the intestinal wall, ultimately affecting bone mineralization2. As a proof-of-concept, we use the described protocol to characterize the biochemical properties of microbial isolates from the femur and tibia of healthy SD rats (Corynebacterium stationis, Staphylococcus cohnii subsp., and Enterococcus faecalis). These microbial isolates were selected as the bone is a closed and hypoxic environment, and characterizing these microbes from bone can be a challenging task. Various articles have detailed the processes used in studying these microbes; however, there are few that provide a complete protocol to identify host-isolated microorganisms.
In establishing the proper culture conditions, the oxygen requirements of the microbe need to be understood via the use of Fluid Thioglycollate Medium (FTM). Microbes with different oxygen requirements form stratified layers in the clear FTM liquid3. Based on the stratification profile, the oxygen requirement of the microbe is then used to investigate the growth of the cells. Microbes that thrive on the surface of the FTM liquid are obligate aerobes, whereas microbes that grow at the bottom are obligate anaerobes. Microbes that grow as a suspension in the FTM liquid are either facultative or aerotolerant anaerobes. The microbial growth rate is established by observing the exponential growth phase of the microbial cells. The growth profile is then compared to the biofilm formation of the microbe. Biofilms are largely composed of multiple species that directly and indirectly affect each other's health. During this process, beneficial interactions among microbial communities select for attachment, providing a spatial structure that favors the evolution of active reciprocal interactions. For example, the co-culture of Paenibacillus amylolyticus and Xanthomonas retroflexus exhibit facultative symbiotic interactions in a static environment, promoting rapid biofilm growth13. Microbes adapt to the host tissues via biofilm formation for sustained localization, protecting themselves against harsh environments and evading the host's immune system4,5,6,7. Biofilms are usually dense and multilayered structures that help microorganisms resist external subcritical stimuli; for example, E. faecalis in dental pulp enhances its resistance to antibiotics by increasing biofilm formation when faced with subconcentrations of tetracycline and vancomycin8.
Chemotaxis enables microorganisms to move according to chemical gradients, and signaling pathways are widely distributed in a variety of pathogenic bacteria. Some pathogenic microorganisms migrate to specific sites, under the guidance of chemical signals, to cause infectious diseases14. For example, Xanthomonas spp. express 19 chemoreceptor and 11 flagellin proteins in the host, triggering bacterial binding and, ultimately, ulceration15. There are also specific proteins in the bacteria (pectin-binding proteins) that guide the specific migration of bacteria to nutrients, which can lead to better growth16. This is also critical for bacteria that may exist in nutrient-poor environments. Microbial cells often rely on chemotactic motility to draw themselves to a conducive growth environment while evading other predatory cells and toxins that harm cellular viability. Building on previously established soft agar approaches to determine the chemotaxis, we develop a diffusible method to generate a chemoattractant gradient for testing the microbial chemotaxis.
This paper describes the methods for determining the growth conditions, biofilm formation, and chemical tropism of bacteria, using Corynebacterium stationis, Staphylacoccus cohnii, Enterococcus faecalis, Staphylacoccus aureus ATCC 25923, and Enterococcus faecalis V583 as examples (see Figure 1). The optimization of microbial growth conditions uses FTM to determine the oxygen requirements of the microbe, while biofilm formation uses glass surfaces as a solid backing, and the biofilm mass is counterstained with crystal violet. The microbe's chemical tropism relies on its chemotactic behavior, where through 3D printing (Supplemental Figure S1), a standardized method is used to generate a chemical reservoir for the chemoattractant in a soft agar matrix (Supplemental Figure S2).
Protocol
Representative Results
Discussion
We isolated and identified five species of bacteria by sequential methods. The growth of bacteria has minimal nutrient requirements: the minimal medium-a medium containing only inorganic salts, a carbon source, and water. Although the bacteria in the experimental group were found on MH solid plates, we used half-dose MH medium to verify the chemotaxis of the bacteria and achieved good results. However, we also performed control experiments using minimal medium. M9 basic medium was used in the experiment (see Tabl…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The development of this technique was supported by the funds from the National Natural Science Foundation of China's Research Fund for International Young Scientists (22050410270), the Shenzhen Special Fund for Innovation and Entrepreneurship of Overseas High-level Talents Peacock Team (KQTD20170810111314625), and the Guangdong Innovative and Entrepreneurial Research Team Program (2019ZT08Y191). We would like to offer our sincere gratitude to Miss Chen Xinyi for her assistance in proofreading the document and laboratory management.
Materials
| Chemical/Solution | |||
| 1% crystal violet dye solution | Solarbio | G1062 | 100 mL |
| Agar | Sigma-Aldrich | V900500 | Used to obtain semi-solid plates, 20 g |
| Centrifuge tube | Corning | 430790 | 15 mL |
| Fluid thioglycollate medium | Kinghunt | K0001 | 29.3 g |
| Mueller Hinton II Broth medium | Solarbio | NO.11865 | 100 g |
| Petri dishes | Bkman | B-SLPYM90-15 | Plastic Petri dishes, circular, 90 mm x 15 mm |
| Potassium Chloride | VETEC | WXBC4493V | 0.2 g |
| Potassium Dihydrogen Phosphate | aladdin | 04-11-7758 | 0.24 g |
| Sodium chloride | Macklin | S805275 | 8.0 g |
| Sodium phosphate dibasic | aladdin | 7558-79-4 | 1.44 g |
| Terrific Broth medium | Solarbio | LA2520 | 200 g |
| Kits/ Equipment | |||
| Anaerobic incubator | Longyue | ||
| Biochemical incubator | Blue pard | LRH-70 | |
| Microplate reader | Spark | ||
| Tanon 5200multi imaging system | Tanon | 5200CE | |
| Thermostatic water bath | Jinghong | DK-S28 |
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