TWiM #315: How Pseudomonas Became a Global Pathogen

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 1:13 minutes

“Background Before the COVID-19 pandemic there has been a constant increase in antimicrobial resistance (AMR) of Escherichia coli, the most common cause of urinary tract infections and bloodstream infections. The aim of this study was to investigate the impact of the COVID-19 pandemic on extended-spectrum β-lactamase (ESBL) production in urine and blood E. coli isolates in Finland to improve our understanding on the source attribution of this major multidrug-resistant pathogen.

Methods Susceptibility test results of 564,233 urine (88.3% from females) and 23,860 blood E. coli isolates (58.8% from females) were obtained from the nationwide surveillance database of Finnish clinical microbiology laboratories. Susceptibility testing was performed according to EUCAST guidelines. We compared ESBL-producing E. coli proportions and incidence before (2018–2019), during (2020–2021), and after (2022) the pandemic and stratified these by age groups and sex.

Results The annual number of urine E. coli isolates tested for antimicrobial susceptibility decreased 23.3% during 2018–2022 whereas the number of blood E. coli isolates increased 1.1%. The annual proportion of ESBL-producing E. coli in urine E. coli isolates decreased 28.7% among males, from 6.9% (average during 2018–2019) to 4.9% in 2022, and 28.7% among females, from 3.0 to 2.1%. In blood E. coli isolates, the proportion decreased 32.9% among males, from 9.3 to 6.2%, and 26.6% among females, from 6.2 to 4.6%. A significant decreasing trend was also observed in most age groups, but risk remained highest among persons aged ≥ 60 years.

Conclusions The reduction in the proportions of ESBL-producing E. coli was comprehensive, covering both specimen types, both sexes, and all age groups, showing that the continuously increasing trends could be reversed. Decrease in international travel and antimicrobial use were likely behind this reduction, suggesting that informing travellers about the risk of multidrug-resistant bacteria, hygiene measures, and appropriate antimicrobial use is crucial in prevention. Evaluation of infection control measures in healthcare settings could be beneficial, especially in long-term care.” (Ilmavirta et al. 2024, no change)

1.2. Main paper; discussion starts at 20:25 minutes

Weimann et al (2024) is not licensed for Creative Commons use, so the abstract cannot be copied here. Please see the article at the journal’s web page.

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)

²Learning Objectives: Lower Order (L) or Higher Order (H)

4. Techniques Described (with Time Stamps)

4.1. Snippet Paper

• Antimicrobial Susceptibility Testing (4:50–5:28): A technique that determines which antibiotics are effective against a specific microorganism causing an infection. This test helps clinicians choose the most appropriate antibiotic treatment and dosage, especially when there’s a concern for antibiotic resistance.
• Finres Database Analysis (6:50–7:22): This database is a nationwide surveillance system of Finnish clinical microbiology laboratories that contains information on antimicrobial susceptibility test results for clinically important bacteria. It has 848,168 urine cultures and 56,788 blood cultures and, in the study, was analyzed for the presence of E.coli.

4.2. Main Paper

• Multi-locus Sequence Typing (30:34–32:00): Multilocus Sequence Typing (MLST) is a molecular technique used to classify bacterial and fungal strains based on the sequences of multiple housekeeping genes. By sequencing these conserved but variable genes, MLST assigns unique allele numbers to each variant and combines them into a sequence type (ST) for precise strain identification. This method is used to track pathogen outbreaks, understand genetic diversity, and compare strains across global databases.
• Bayesian temporal reconstruction and skyline demographic modeling (32:30–34:17): Statistical methods used to reconstruct the history of changes in a population. These are very specialized techniques used by molecular evolutionary biologists to study evolutionary histories of a population. Here, they are used to infer that the pathogen P. aeruginosa clones emerged between the late 17th and late 20th century, and that this population underwent at least one major population expansion between the years 1850 and 2000.
• Panaroo (29:45–50:26): Panaroo is a system for pan-genome analysis. It compares genes that are present in core, accessory, and unique gnomes to reveal host specific adaptations and antimicrobial resistance mechanisms. This allowed the researchers to find that there was an enrichment in genes related to transcriptional regulation, inorganic ion transport in lipid metabolism, and protein turnover.

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

5.1. Snippet Paper

• Antibiotic Resistance (1:13–5:28, 17:12–18:14): Bacteria develop the ability to resist the effects of antibiotics, meaning the drugs are no longer effective in killing or slowing down the bacteria’s growth. This resistance arises from bacterial mutations and the selection of resistant bacteria during antibiotic treatment, leading to infections that can be difficult or impossible to treat. They discuss examination of extended-spectrum beta-lactamase (ESBL)-producing E. coli, which resist multiple beta-lactam antibiotics, and discussion of antimicrobial stewardship programs to reduce resistance.
• Human Microbiome (9:24–9:42): All the microbiota that reside on or within human tissues and biofluids, along with the corresponding anatomical sites in which they reside. In the podcast, they discuss E. coli in the urinary tract and its colonization due to microbiome imbalances, and increased risk in diabetics due to sugar availability in urine.
• Opportunistic Infections (12:32–14:13): A microorganism that does not typically cause disease in a healthy individual, but can cause illness or infection when the host’s immune system is weakened or compromised. In the podcast, there is discussion of urinary tract infections (E. coli infections), factors that contribute to susceptibility, and changes in infection rates due to altered behaviors during the pandemic.
• Antibiotics Targeting DNA Replication (15:02–16:18): They discuss fluoroquinolones, a class of broad-spectrum antibiotics that target bacterial DNA replication. Their overuse during COVID-19 has contributed to resistance.

5.2. Main Paper

• Horizontal Gene Transfer (20:25–29:43): The movement of genetic material between organisms that are not in a parent-offspring relationship. They discuss an explanation of how Pseudomonas aeruginosa acquires virulence factors through horizontal gene transfer, leading to epidemic clones.
• Pathogenicity Islands (20:25–29:43): Distinct regions of DNA found in the genomes of many pathogenic bacteria, often containing clusters of genes responsible for virulence. Identification of pathogenicity islands in Pseudomonas aeruginosa, including those responsible for Type III secretion systems that enhance virulence was discussed.
• Microbial Genetics and Gene Expression (29:43–50:26): The use of genomic sequencing and transcriptomics to analyze gene expression changes associated with host adaptation. In this study, it was in Pseudomonas aeruginosa.
• Virulence Factors (50:26–55:45): Molecules produced by a pathogen (like bacteria, viruses, or fungi) that enhance its ability to cause disease by enabling it to infect, colonize, and damage a host. These factors play a crucial role in the pathogen’s ability to overcome the host’s defense mechanisms and establish an infection. Identification of key virulence mechanisms that enable Pseudomonas aeruginosa to survive within macrophages and cause chronic infections in cystic fibrosis patients was studied.
• Innate Immunity (50:26–56:06): The body’s natural, non-specific defense system present from birth, providing immediate protection against a wide range of pathogens. It’s the first line of defense and doesn’t require prior exposure to a specific threat. In the study, they analyze how Pseudomonas aeruginosa survives within macrophages by evading innate immune responses, specifically related to neutrophils and macrophages.
• Epidemiology (56:06–57:29): The study of how diseases and other health-related conditions are distributed in populations and the factors that influence their occurrence. It aims to understand the causes of diseases, identify risk factors, and develop strategies for prevention and control. There was discussion on the need for surveillance of epidemic bacterial clones, public health measures to prevent cross-infections, and global efforts to track bacterial evolution.

6. Podcast Questions

1. Which of the following best describes the function of extended spectrum beta-lactamase (ESBL) in E. coli?
   1. It increases the permeability of the bacterial cell wall to beta-lactam antibiotics.
   2. It facilitates the uptake of antibiotics into the bacterial cytoplasm.
   3. It inactivates antibiotics by hydrolyzing their beta-lactam ring structure.
   4. It enhances immune recognition by modifying surface antigens.
2. Which of the following led to a decline in ESBL-producing E. coli in Finland during the COVID-19 pandemic according to the author’s conclusions?
   1. Implementation of mandatory mask wearing policies
   2. Reduction in international travel
   3. Development of novel antibiotics effective against ESBL-producing E. coli
   4. Increased public awareness of the dangers of antibiotic overuse
3. Which strategy is recommended to combat the rise of ESBL-producing E. coli infections during global health crises?
   1. Reducing antibiotic usage in agriculture
   2. Enhancing surveillance and control measures
   3. Developing novel antibiotics
   4. Promoting alternative medicine practices
4. Why might blood infections with ESBL-producing E. coli show different trends than urinary infections during the pandemic?
   1. Blood infections were typically community-acquired and therefore decreased more.
   2. Patients with blood infections were less likely to seek hospital care during the pandemic.
   3. Blood infections often required hospitalization, so sampling rates remained more stable.
   4. Blood infections were not influenced by travel-related bacterial exposure.
5. Based on the interpretations of the trends described in the podcast, finish the sentence to identify the trends that you would expect to see after the pandemic. ESBL-producing E. coli would appear more frequently in ____________________ (pick all that apply).
   1. Populations who work outside the home for extended periods of time.
   2. All populations once the lock down was lifted.
   3. Populations who use public transportation like airplanes.
   4. Populations who drink a lot of water and urinate often.
6. Which of the following best describes “saltatory evolution” in the context of P. aeruginosa evolution?
   1. Gradual accumulation of point mutations
   2. A slow, continuous change due to selection pressure
   3. Sudden genetic shifts due to horizontal gene transfer
   4. Stabilizing selection to preserve ancient traits
7. What does the study’s use of pan-genome analysis tell us about P. aeruginosa clones?
   1. It reveals the full set of genes present in every Pseudomonas aeruginosa strain, identifying universal characteristics.
   2. It identifies the evolutionary pressures that drive the loss of genetic material in epidemic clones.
   3. It shows the collection of essential, accessory, and strain-specific genes that define the pathogenicity and adaptability of each clone.
   4. It helps pinpoint the exact mutation in the P. aeruginosa genome responsible for causing cystic fibrosis in patients.
8. Which method was used to determine the sequence types of P. aeruginosa clones?
   1. CRISPR-Cas9 gene editing
   2. Multi-locus sequence typing (MLST)
   3. Skyline demographic modeling
   4. 16s rDNA sequence alignment only
9. Which regulatory gene was found to be upregulated in epidemic P. aeruginosa clones infecting CF patients, enhancing their intracellular survival?
   1. lacZ
   2. recA
   3. rpoB
   4. dksA1
10. Which of the following experimental findings most strongly supports the hypothesis that transcriptional regulation is involved in the adaptation of P. aeruginosa clones to human hosts?
    1. Mutants with deleted virulence genes showed a significantly higher survival rate in macrophages.
    2. Increased survival within macrophages was observed in high-affinity CF clones, associated with a specific transcription factor, Dksa1.
    3. Pan-genome analysis identified genes exclusive to environmental isolates, which were absent in clinical isolates.
    4.  Clones with higher mutational burdens showed a reduced ability to survive in macrophages, regardless of host type.

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 1ABC).

7.1. First Figure Reading Exercise

7.1.2. Questions

1. What color line represents data for the ESBL+ blood cultures in males and what relative decrease (pre- to post-pandemic) was observed for this group and bacterial source?
   1.  red; 32.9%
   2. gold; 28.7%
   3. peach; 26.6%
   4. pale gold; 28.7%
2. Which of the following statements describes the overall trend in extended-spectrum beta-lactamases (ESBL)-producing E. coli between 2018 and 2022?
   1. Proportions of ESBL-producing E. coli increased linearly across all groups regardless of sex or sample.
   2. Both groups decreased, but females decreased more significantly than men across both isolates.
   3. Both groups decreased, but males decreased more significantly than females across both isolates.
   4. There was no change in proportion of ESBL-producing E. coli between 2018 and 2022 in Finland.
3. What do the data suggest about the impact of the COVID-19 pandemic on ESBL-producing E. coli prevalence in Finland between 2018 and 2022?
   1. ESBL-producing E. coli increased rapidly due to disruptions in healthcare and surveillance.
   2. The COVID-19 pandemic had no effect on ESBL-producing E. coli prevalence in Finland.
   3. There was an initial decline, but it increased back to the baseline post-pandemic.
   4. The pandemic decreased the rising prevalence of ESBL-producing E. coli in Finland.
4. Suppose a similar study in another country shows no change in ESBL-producing E. coli rates over the same period. What might be a reasonable explanation for the difference from Finland’s trends?
   1. The other country may have used a different method to calculate antimicrobial-associated disease rates.
   2. The other country may not have enforced travel limits or antibiotic use policies during the pandemic.
   3. The other country may have started with fewer infections, reducing the chance of a noticeable change.
   4. Finland’s results may reflect sampling bias or errors in how the data were collected or reported.

7.2. Second Figure Reading Exercise

7.2.2. Questions

1. The geographical location of P. aeruginosa clones in this study are identified on the map (panel A).  What feature indicates the abundance of clones found at each location?
   1. circle color
   2. circle map location
   3. circle size
   4. circle transparency
2. Which of the following best describes the relationship between P. aeruginosa clones used in this study and their geographical distribution (panel A)?
   1. Clones are evenly distributed across all seven continents.
   2. Clones show regional clustering, with some clones found globally.
   3. Clones are restricted to clinical settings in Europe and North America only.
   4. Clones are more globally found and evenly spread except for Antarctica.
3. What does the red color in Panel B indicate about a P. aeruginosa clone?
   1. It has caused hospital-acquired infections.
   2. It has fewer than 30 isolates associated with it.
   3. It has been classified as an epidemic clone.
   4. It was isolated only from the natural environment.
4. Which clone is the most prevalent and globally distributed of those in this study (panel C)?
   1. ST111
   2. ST27
   3. ST309
   4. ST235

8. Paper Information and Licensing

8.1. Snippet paper

• Ilmavirta H, Ollgren J, Räisänen K, Kinnunen T, Hakanen AJ, Rantakokko-Jalava K, Jalava J, Lyytikäinen O. 2024. Impact of the COVID-19 pandemic on extended-spectrum β-lactamase producing Escherichia coli in urinary tract and bloodstream infections: results from a nationwide surveillance network, Finland, 2018 to 2022. Antimicrob Resist Infect Control. 13(1):72. doi: 10.1186/s13756-024-01427-z.
• 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://aricjournal.biomedcentral.com/articles/10.1186/s13756-024-01427-z .

8.2. Main paper

• Weimann A, Dinan AM, Ruis C, Bernut A, Pont S, et al.. 2024. Evolution and host-specific adaptation of Pseudomonas aeruginosa. Science. 385(6704):eadi0908. doi: 10.1126/science.adi0908.
• 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 website.