TWiM #263: Lavender and Catheters

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 3:41 minutes

Abstract

• This article is not licensed for Creative Commons use; see licensing information. Thus the abstract and figures cannot be copied here. Please see the article at the journal’s web page. https://doi.org/10.1111/lam.13681

1.2. Main paper; discussion starts at 32:22 minutes

“Microbial adhesion to medical devices is common for hospital-acquired infections, particularly for urinary catheters. If not properly treated these infections cause complications and exacerbate antimicrobial resistance. Catheter use elicits bladder inflammation, releasing host serum proteins, including fibrinogen (Fg), into the bladder, which deposit on the urinary catheter. Enterococcus faecalis uses Fg as a scaffold to bind and persist in the bladder despite antibiotic treatments. Inhibition of Fg–pathogen interaction significantly reduces infection. Here, we show deposited Fg is advantageous for uropathogens E. faecalis, Escherichia coli, Pseudomonas aeruginosa, K. pneumoniae, A. baumannii, and C. albicans, suggesting that targeting catheter protein deposition may reduce colonization creating an effective intervention for catheter-associated urinary tract infections (CAUTIs). In a mouse model of CAUTI, host-protein deposition was reduced, using liquid-infused silicone catheters, resulting in decreased colonization on catheters, in bladders, and dissemination in vivo. Furthermore, proteomics revealed a significant decrease in deposition of host-secreted proteins on liquid-infused catheter surfaces. Our findings suggest targeting microbial-binding scaffolds may be an effective antibiotic-sparing intervention for use against CAUTIs and other medical device infections.” (Andersen et al. 2022, no changes)

2. Vision and Change Core Concepts and 2024 ASM Fundamental Statements

3.  Potential Learning Objectives for the Podcast

¹Papers: Snippet (S) or Main (M)
²Learning Objectives: Lower Order or Higher Order (H)

4. Techniques Described (with Time Stamps)

Here is a link to a bio-dictionary that has many, but not all definitions if you need a definition: Explore Biology Bio-Dictionary

4.1. Snippet Paper

• Plate Count Techniques (7:07–7:09): These are conventional bacterial quantification techniques and relies on culturing of colonies on a plate, which is then counted. Dilutions are usually needed to produce countable plates.
• Optical Density (OD) Studies (10:50–11:00): These are assays that use light scatter to quantify the opaqueness of a solution.  Here, optical density measurements were used to determine level of disruption of biofilms grown on microscope slides. Higher transmission of light is evident with increased biofilm disruption.
• Minimal Inhibitory Concentration (MIC) Assays (11:19–12:00): assays used to determine the minimal concentration of an antimicrobial compound that produces visible reduction in bacterial growth. Dilutions are used to explore concentration gradients.
• XTT Assay (16:40–18:10): This is an assay that uses Sodium 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide, commonly known as XTT, to determine the metabolic activity of a bacterial culture. These measurements can serve as indicators of cell viability and population growth. The assay uses the XTT indicator, which measures cellular redox potential. The color indicator changes from clear to deep red, so a spectrophotometer can be used to quantify metabolic activity.
• Propidium Monoazide (PMA) PCR assay (20:45–21:51): This is an assay that uses a light-sensitive DNA binding agent (PMA) that is cell-membrane impermeable. When exposed to light, it forms a covalent bond with DNA, preventing amplification of the DNA by PCR. Only dead cells can take up the dye, and viable cells can be quantified by PCR amplification of marker genes like 16S rDNA.
• Confocal Laser Scanning Microscopy (CLSM) (25:15–25:45): This is a specialized microscope technique that uses point-illumination to light up blurry or unfocused background areas in a sample, producing a high-resolution image. It allows the user to control the depth of field viewed, and multiple 2-D images can be obtained and used to generate a detailed 3-D rendering of the object being viewed.

4.2. Main Paper

• Fluorescence Microscopy (43:46): This is a microscopy technique that uses a changeable wavelength light source to excite fluorescence dyes to localize a target.
• in vivo CAUTI Mouse Model (54:15–55:04): This is a mouse model system that uses catheter bladder implants to study bacterial colonization in UTIs in mice.

5. Connections to General Microbiology Processes/Concepts (with Time Stamps)

5.1. Snippet Paper

• Listeria monocytogenes (4:52–5:10): An important foodborne pathogen that grows well in refrigeration temperatures, can form problematic biofilms and can also enter a Viable but Non-Culturable (VBNC) state.
• Viable but Non-Culturable (VBNC) State (5:08–7:08): Cells can enter this Viable but Non-Culturable state. If cells are unable to grow on bacteriological media, we cannot use cultural methods to detect them.
• Biofilms (7:08–10:09; 15:05–29:30): Bacteria form complex communities that are attached to a surface, and these structures are difficult to disrupt and provide environments where resistance against bacterial control agents like common disinfectants and antibiotics can develop.
• Psychrophiles (8:17–10:09): Some bacteria thrive at low temperatures, including those like Listeria, which then pose a risk as they readily grow in foods stored in the refrigerator.
• Redox reactions (17:34–19:20): Metabolically active cells move electrons during metabolism, and this can be used in assays using redox dyes.

5.2. Main Paper

• Biofilms, Antibiotic resistance, and the Immune System (36:14–37:09): Around 70% of bacteria spend at least part of their life in biofilms which are formed on surfaces. Cells in these biofilms can become resistant to antibiotics, and these structures also evade our immune system, making biofilms especially problematic when they exist in a patient.
• Inflammatory Response (38:19–39:15; 48:00–59:12): Catheters increase inflammation of the bladder, promoting production of fibrinogen. Fibrinogen is a great substrate for bacterial attachment and colonization.
• Bacterial Adhesins (39:07–40:00): These are proteins that allow bacteria to attach to surfaces and can be virulence factors, as they foster colonization.

6. Podcast Questions

1. Which of the following statements is the best description of a bacterial biofilm?
   1. A complex microbial community that attach to surfaces and are stabilized by a matrix of proteins and polysaccharides.
   2. A cluster of cells in circulation that evade the immune response by attaching to each other.
   3. Secreted lipopolysaccharides produced independently by free floating bacteria.
2. Bacteria that can survive or grow in low temperatures (typically −20°C to 15°C) are called:
   1. mesophiles
   2. halophiles
   3. thermophiles
   4. psychrophiles
3. Which of the following contribute to the risk of contamination with Listeria monocytogenes during the processing of ready-to-eat meals? [Select all that apply.]
   1. Listeria monocytogenes can survive low temperatures (0–10°C)
   2. Listeria monocytogenes can enter a viable but not culturable state.
   3. Listeria monocytogenes can form dormant spores.
   4. Listeria monocytogenes produces endotoxins.
4. How might viable but non culturable (VBNC) bacteria create a concern for food safety?
   1. Quantitating bacteria with culture based methods will overestimate the number of bacteria that are actually present.
   2. Quantitating bacteria with culture based methods will underestimate the number of bacteria that are actually present.
   3. There are no methods for quantitating viable but non culturable bacteria.
   4. As long as disinfection protocols are followed, there is no cause for concern.
5. Fibrinogen is a blood protein that:
   1. naturally prevents bacterial growth.
   2. can provide a surface for bacterial attachment.
   3. is inhibited in an inflammatory response.
6. What percentage of hospital acquired infections are due to catheter-associated urinary tract infections (CAUTI)?
   1. 5%
   2. 15%
   3. 40%
   4. 75%
7. Place the following items in a sequence from insertion of the catheter to development of a urinary tract infection: (Sse numbers 1, 2, 3)
   1. _____ production of fibrinogen
   2. _____ attachment and growth of pathogens
   3. _____ increase inflammation of the bladder

7. Figure Reading Exercises

The following are three figure reading exercises, one from the snippet paper (Figure 1) and one from the main paper (Figures 2A-F).

7.1. First Figure Reading Exercise

7.1.2. Questions

1.  What treatment is noted by the pattern that is a white background and tiny black dots?
   1. hydrogen peroxide
   2. lactic acid
   3. lavender essential oil
   4. sodium hypochlorite
2. The data using the minimum inhibitory concentration (MIC, this is the 1MIC level) for each disinfectant with treatment at 32^oC is shown in panel a.  Which treatments are significantly different than sodium hypochlorite (bleach)? [pick all that apply]
   1. hydrogen peroxide
   2. lactic acid
   3. lavender essential oil
   4. control
3. Which disinfectant was the least effective at disrupting biofilms at 32^oC (at most MIC)?
   1. lactic acid
   2. lavender essential oil
   3. sodium hypochlorite
   4. hydrogen peroxide
4. At which MIC and temperature is lavender essential oil the single most effective disinfectant for disrupting biofilms? [pick all that apply]
   1. 1/2 MIC at 32°C
   2. MIC at 32°C
   3. 2 MIC at 32°C
   4. 1/2 MIC at 10°C
   5. MIC at 10°C
   6. 2 MIC at 10°C
5. Based on the data, how does reducing the temperature generally impact lavender oil’s ability disrupt established Listeria biofilms?
   1. High temperature reduces disinfectant effectiveness to the level of the other disinfectants.
   2. Low temperature reduces disinfectant effectiveness to the level of the other disinfectants.
   3. High temperature reduces effectiveness to the level of the other disinfectants at high MIC only.
   4. Low temperature reduces effectiveness to the level of the other disinfectants at high MIC only.

7.2. Second Figure Reading Exercise

Experimental Background (Andersen et al., Figure 2A-F)

Medical devices can add quality of life and enhance treatments, but they can also contribute to infection.  In particular, they can promote biofilm formation, which are complex microbial communities that are more resistant to antimicrobial treatments than free-floating microbes.  Biofilms can form on catheters, resulting in catheter-associated urinary tract infections (CAUTIs) which account for 40% of hospital acquired infections that easily disseminate and cause about 30% mortality.  In this study, Andersen et al. (2022) investigate how host components contribute to biofilm formation using common uropathogenic bacteria: Enterobacter. faecalis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella. pneumoniae, Acinetobacter baumannii, and Candida albicans.  The host protein of interest in this particular experiment is fibrinogen (Fg) because it was previously determined that E. faecalis can use this protein as a biofilm scaffold.  Additionally, Fg is produced in the bladder as a result of inflammation due to catheterization. To determine which of the other bacteria could also utilize this mechanism, the researchers treated silicone catheters with fibrinogen, bovine serum albumin (BSA, a common serum protein), or no coating (uncoated; UC).  The catheters were inserted into mouse bladders which were then inoculated with each bacterial species and subsequently quantified using immunofluorescence.  The data for each is presented as a bar graph where the binding of pathogen to fibrinogen is set to 100% to allow for the comparison to other treatments.

7.2.2. Questions

1.  Controls are part of any good experiment, and here we have two controls, untreated and BSA-treated silicone.  What does the BSA-treated control tell us?
   1. As a control, it tells us the level of bacteria binding in the presence of a common body protein.
   2. As a positive control, it tells us the bacteria are capable of binding the silicone tubing.
   3. As a negative control, it tells us the bacteria are not capable of binding silicone tubing.
   4. As a control, it tells us how many bacteria survive the culture and quantitation procedures.
2. In this experiment, the researchers have shown data for a bacterial positive control, one that they knew should be impacted by fibrinogen. Which species is the positive control?
   1. E. faecalis
   2. E. coli
   3. P. aeruginosa
   4. K. pneumoniae
   5. A. baumannii
   6. C. albicans
3. Bacteria adhering to the silicone catheter were quantified using immunofluorescence.  The data for each species are displayed by bar graphs. Match the graph feature with its description. (1 = mean; 2 = median; 3 = standard deviation; 4 = standard error of the mean; 5 = full data range excluding outliers; 6 = statistically significant; 7 = not statistically significant)
   1. ________ bar height
   2. ________ whiskers
   3. ________ asterisks
   4. ________ ns
4. Which microbial pathogens bind better to silicone catheters treated with fibrinogen compared to the controls? [pick all that apply]
   1. E. faecalis
   2. E. coli
   3. P. aeruginosa
   4. K. pneumoniae
   5. A. baumannii
   6. C. albicans
5. Which microbial pathogens bind better to silicone catheters treated with BSA compared to the untreated control? [pick all that apply]
   1. E. faecalis
   2. E. coli
   3. P. aeruginosa
   4. K. pneumoniae
   5. A. baumannii
   6. C. albicans
6. Which bacterial binding was impacted the most by adding the fibrinogen coating to the catheter?
   1. E. faecalis
   2. E. coli
   3. P. aeruginosa
   4. K. pneumoniae
   5. A. baumannii
   6. C. albicans
7. Which bacterial binding was impacted the least by adding the fibrinogen coating to the catheter?
   1. E. faecalis
   2. E. coli
   3. P. aeruginosa
   4. K. pneumoniae
   5. A. baumannii
   6. C. albicans
8. In a hypothetical follow up experiment, the researchers incubated fibrinogen-coated catheter tubing with either uropathogenic E. faecalis alone (monoculture) or with a mixture of uropathogenic E. faecalis and Finegoldia magna, a bacterium that produces SufA, a fibrinogen-degrading protease. In this experiment, you would expect to see _________.
   1. enhanced binding of E. faecalis in the mixed culture, but not in the monoculture
   2. decreased binding of E. faecalis in the mixed culture, but not in the monoculture
   3. no difference in binding levels for E. faecalis between the two treatment groups
   4. enhanced binding of E. faecalis and Finegoldia magna as they form a biofilm

8. Paper Information and Licensing

8.1. Snippet paper

• Han X, Chen Q, Zhang X, Peng J, Zhang M, Zhong Q. 2022. The elimination effects of lavender essential oil on Listeria monocytogenes biofilms developed at different temperatures and the induction of VBNC state. Lett Appl Microbiol. 74:(6)1016-1026. https://doi.org/10.1111/lam.13681.
• This article is not licensed for Creative Commons use. Thus, the abstract and figures cannot be copied here. See the article on the journal’s web page.

8.2. Main paper

• Andersen M, Fong C, La Bella A, Molina J, Molesan A, Champion M, Howell C, Flores-Mireles A. 2022. Inhibiting host-protein deposition on urinary catheters reduces associated urinary tract infections. eLife 11:e75798, https://doi.org/10.7554/eLife.75798
• This article is licensed for Creative Commons use using CC BY 4.0, which allows re-use and adaptation with proper attribution and notation of any changes; see https://doi.org/10.7554/eLife.75798