TWiM #165: Pumping Copper

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 3:01 minutes

“Background: Athletic centers have been locations for the transmission of community-acquired infections. This study assessed the capacity of copper alloys to reduce the bacterial burden associated with high-touch athletic center equipment. Copper alloy weights and grips were rotated with rubber coated and stainless steel controls in an undergraduate college athletic center over a 16-month period. The athletic center is used by college athletic teams, student body, and local community. Methods: The primary outcome was to compare bacterial burdens on copper and control grips by swabbing surfaces. Significance was determined using the nonparametric Mann-Whitney U test with significance assessed at P < .05. Secondary outcomes included characterizing bacterial communities on surfaces and conducting antibiotic susceptibility testing using the Kirby-Bauer disk diffusion method. Results: Control athletic center components carried bacterial loads 94% larger than those found on copper alloy components. Bacterial community characterization revealed Staphylococcus to be the most common bacterial genus found on grip surfaces. Antibiotic resistance testing of the Staphylococcus isolates revealed that all isolates were susceptible to vancomycin and linezolid, whereas 35% of copper alloy isolates and 44% of control isolates were resistant to erythromycin. Conclusions: Copper alloys can mitigate the bacterial burden on high-touch surfaces. Strategically placing copper alloys in areas of high human contact can augment infection control efforts and potentially decrease community-acquired infections in athletic centers.” (Ibrahim et al. 2018)

1.2. Main paper; discussion starts at 22:00 minutes

“Microbial pathogens commonly escape the human immune system by varying surface proteins. We investigated the mechanisms used for that purpose by Pneumocystis jirovecii. This uncultivable fungus is an obligate pulmonary pathogen that in immunocompromised individuals causes pneumonia, a major life-threatening infection. Long-read PacBio sequencing was used to assemble a core of subtelomeres of a single P. jirovecii strain from a bronchoalveolar lavage fluid specimen from a single patient. A total of 113 genes encoding surface proteins were identified, including 28 pseudogenes. These genes formed a subtelomeric gene superfamily, which included five families encoding adhesive glycosylphosphatidylinositol (GPI)-anchored glycoproteins and one family encoding excreted glycoproteins. Numerical analyses suggested that diversification of the glycoproteins relies on mosaic genes created by ectopic recombination and occurs only within each family. DNA motifs suggested that all genes are expressed independently, except those of the family encoding the most abundant surface glycoproteins, which are subject to mutually exclusive expression. PCR analyses showed that exchange of the expressed gene of the latter family occurs frequently, possibly favored by the location of the genes proximal to the telomere because this allows concomitant telomere exchange. Our observations suggest that (i) the P. jirovecii cell surface is made of a complex mixture of different surface proteins, with a majority of a single isoform of the most abundant glycoprotein, (ii) genetic mosaicism within each family ensures variation of the glycoproteins, and (iii) the strategy of the fungus consists of the continuous production of new subpopulations composed of cells that are antigenically different.” (Schmid-Siegert et al. 2017)

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

3.  Potential Learning Objectives for the Podcast

¹ Papers: Snippet (S) or Main (M)

² Learning 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

• Antibiotic Susceptibility Testing (13:55–14:18): These are assays used to determine the sensitivity of a microbe to antimicrobial compounds.  Here, it  used to test the bacteria found on the tested surfaces for resistance against vancomycin, erythromycin, and linezolid.
• Selective Culturing (15:55–17:00): This is a method for reducing background microbes in a culture.  Here, the researchers were interested in clinical spread of C. difficile by spores so they pre-sanitized with alcohol to reduce background microbes because C. difficile spores are resistant to alcohol.

4.2. Main Paper

• Whole Genome Sequencing (27:30): This is a sequencing and bioinformatics method to fully sequence and assemble entire genomes.  Here, it was used to compare the similarities and differences of Pneumocystis pneumonia infecting different animals (human, rat, mouse) because each strain is host-specific.
• PacBio Sequencing (27:35): This is a “next next generation” sequencing technology that produces long reads of DNA rather than short reads.   This makes it easier to confidently assemble genomes with repetitive DNA  elements such as this fungus has.
• Polymerase Chain Reaction (PCR) (27:25): Polymerase chain reaction (PCR) is a molecular detection method using primers, heat-stable DNA polymerase and a DNA template.  Here, it was used to generate a genomic library of Pneumocystis jirovecii and identify glyocoprotein encoding  genes produced by recombination.

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

5.1. Snippet Paper

5.2. Main Paper

1. What is the criteria that determine whether a copper alloy is antimicrobial?
   1. There is a 99.0% reduction in desiccated spores within 30 minutes.
   2. There is a 99.5% reduction in gram negative bacteria by 1 hour.
   3. There is a 99.9% reduction in all microbes within 2 hours.
   4. There is a 90.5% reduction in fungal spores within 10 hours.
2. Staphylococcus was found to be very abundant and the podcasters were not surprised by this finding. Why?
   1. Staphylococcus is part of the skin microbiome.
   2. Staphylococcus is a common soil bacterium.
   3. Staphylococcus is a waterborne fecal bacterium.
   4. Staphylococcus is part of the “fecal patina.”
3. What was the conclusion of the antimicrobial gym study?
   1. Copper-aluminum alloys are effective for reducing bacterial contamination of gym contact surfaces.
   2. Nickel-zinc alloys are effective for reducing bacterial contamination of gym contact surfaces.
   3. Zinc-aluminum alloys are effective for reducing bacterial contamination of gym contact surfaces.
   4. Copper-nickel alloys are effective for reducing bacterial contamination of gym contact surfaces.
4. Built environments are human-made structures such as buildings.  These necessarily harbor different bacteria than natural environments. Based on this study, which one specific equipment would you recommend replacing with (or embedding) copper alloy to help prevent pathogen spread at the gym in a cost-effective way. Why?
   1. Yoga mats since the contact time is very high and contamination increased for spongy materials.
   2. Bars on the low weight dumbbells since they are used often and were most contaminated.
   3. Stationary bike seats since the contact time is very high and they were moderately contaminated.
   4. The cardio machine handles since they are often sweated on and were highly contaminated.
5. What is pathogen-based antigenic variation?
   1. This is the ability of the pathogen to alter its genome to express different surface markers and evade immune recognition.
   2. This is the ability of the pathogen to destroy host tissues to release different surface markers and evade immune recognition.
   3. This is the ability of the pathogen to alter immune cell function and escape by enhancing recognition of other pathogens.
   4. This is the ability of the pathogen to alter its genome to express host self surface markers and trigger immune tolerance.
6. What features in which msg group’s genetic sequence suggested this fungus uses antigenic variation?
   1. A DNA sequence with no translatable protein and four promoters in Msg-V
   2. A gene lacking a start codon with an antigen trap domain at the end of Msg-V
   3. A promoterless gene with a recombination element at the end of Msg-I
   4. A kinase-deficient coding sequence with a transposon at the end of msg-I
7. If P. jirovecii lost the ability to have antigenic variation, would it make it easier or harder to make a vaccine against this pathogen? What is your reasoning?
   1. Antigenic variation allows the pathogen to deflect immune memory towards other pathogens in the host. So its loss would not affect vaccine development.
   2. Antigenic variation allows the pathogen to continually re-disguise itself from the immune system.  So it’s loss would make it easier to form a successful vaccine.
   3. Antigenic variation causes the pathogen to decrease its replication rate and fungal burden in tissues. So its loss would make it harder to form a successful vaccine.
   4. Antigenic variation allows the pathogen to increase inflammation and chemoattractants during infection. So its loss would make it easier to form a successful vaccine.
8. Why might sequencing using short read next generation technologies hinder finding other antigenic variation systems?
   1. Short sequences won’t allow researchers to detect single nucleotide changes, which are characteristic of recently evolved pathogen genes.
   2. Short sequences won’t allow researchers to detect metabolic pathways, which are characteristic of contingency gene families.
   3. Short sequences won’t allow researchers to detect transcription factors, which are characteristic of gene regulation variation systems.
   4. Short sequences won’t allow researchers to detect repeated genome elements, which are characteristic of antigenic variation genes.

7. Figure Reading Exercises

The following are two figure reading exercises, one from the snippet paper (Figure 1) and one from the main paper (Figure 2).

7.1. First Figure Reading Exercise

Experimental Background (Ibrahim et. al, Figure 1)

The World Health Organization (WHO) has the rise of antibiotic-resistant microbes as a top ten health concern.  Hospital-acquired infections have been reduced by using preventative measures and education.  Community-acquired infections might also be reduced if these were also used more broadly.  Ibrahim et al. (2018) were specifically interested in testing a non-drug alternative preventative measure that has had success in hospitals, copper alloy surfaces.  To get an idea of the microbial contamination in the built environment of a gym, as well as whether copper alloys might inactivate bacteria, they quantified microbial contamination on common high-touch gym equipment.  The gym equipment was constructed with traditional materials (rubber or stainless steel) or a copper alloy and bacteria isolated over a three month period.  They swabbed equipment and grew bacteria; they report their data as colony forming units (CFU) per 100 centimeter square, which are the  y-axis units for this bar chart.

7.1.2. Questions

1. Match the feature with its description.

2. Good experimental design includes controls.  What control condition was included in this experiment? What was the purpose of using this as a control?
   1. Standard grips are the control.  They show us the normal level of bacteria so we can identify whether the copper alloy has any effect.
   2. The low row attachment is the control.  This little used piece of equipment shows us the lowest expected  bacterial contamination.
   3. The dumbbell 15 is the control.  This highly used piece of equipment will show us how high of a contamination to expect.
   4. Continuous cleaning with wipes is the control.  This shows us the effect of standard gym cleaning wipes on bacterial contamination level.
3. What notation tells you the comparison being made is statistically significant, that is truly different? Which comparisons are truly different?
   1. The figure legend notes statistical significance at the P<0.05 level for all dumbbell equipment compared to attachments.
   2. The figure legend notes statistical significance at the P<0.0001 level for copper vs control for each piece of equipment.
   3. The figure legend notes statistical significance at the P<0.001 level for different equipment and also for copper vs. control.
   4. The figure legend notes statistical significance at the P<0.0001 level for dumbbell and kettlebell controls compared to copper.
4. Which piece of equipment with standard grips was most contaminated?  What is your evidence?
   1. Dumbbell 20 pounds, it has the highest median bacterial level.
   2. Dumbbell 15 pounds, it has the highest mean bacterial level.
   3. Dumbbell 20 pounds, it has the most reduction with the copper alloy.
   4. All are contaminated, but we can’t tell which without statistical tests.
5. Does the copper alloy have an effect on bacterial contamination? What is your evidence?
   1. Yes; the copper alloy median (triangle in the blue bar) is less than the standard median (triangle in the gray bar) for each piece of equipment.
   2. No; the blue bar (copper alloy) is approximately equal to the gray bar (standard) for each piece of equipment even though the medians differ.
   3. Yes; the blue bar (copper alloy) is less than the gray bar (standard) for each piece of equipment and that difference is statistically significant.
   4. No; the blue bar (copper alloy) is greater than the gray bar (standard) for each piece of equipment and that difference is statistically significant.

7.2. Second Figure Reading Exercise

7.2.2. Questions