Course:PATH417:2022W2/Case3

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To celebrate Tom’s retirement his wife and two adult children accompany him on a long anticipated cruise. Tom’s asthma flares up a few days before the cruise but with a corticosteroid nebulizer he feels well enough to join the cruise. Even more than the rest of his family, Tom enjoys the various hot tubs aboard the massive ship those first few days, relishing the relaxation after a busy final year at work. On the fifth day of the cruise, Tom wakes up in a sweat with a cough that continues throughout the day. As the day wears on he feels worse with a headache, muscle aches and nausea accompanying the cough. His wife arranges for the cruise doctor to visit him in his cabin. The doctor examines Tom, notes his high temperature, nonproductive cough and recent history of asthma and corticosteroid therapy. She takes a full history including taking note of his activities during the first days of the cruise and diagnoses Tom with pneumonia. She starts Tom on azithromycin. By the time the ship returns to port two days later, 5 more people have been diagnosed with a similar pneumonia, several of whom have a slightly compromised immune system, as Tom does. One of the others is admitted to hospital, where sputum and urine samples are tested and reveal a diagnosis of Legionellosis. Public health authorities are notified and the ship takes extra time in port to allow for an enhanced cleaning to be performed on all of the hot tubs.


1. The Body System

(i) Describe the signs (objective characteristics usually detected by a healthcare professional) and symptoms (subjective characteristics experienced by the patient). Are there any other signs or symptoms that could have been commented on but are not presented in the case? What are the key History of Presenting Illness elements presented?

There are two types of Legionellosis: Legionnaires’ disease, which is the pneumonic form that Tom likely has, and Pontiac fever, which is the non-pneumonic form (1) and is also self-limiting (2). Legionnaires’ disease also results in more severe symptoms, while Pontiac fever results in less severe symptoms (3).

Signs

There are generally no clinical signs that are specific to or characteristic of Legionella infections.  

Symptoms

Symptoms of Legionella infection that were experienced by Tom include high temperature (i.e. fever), a non-productive (dry) cough, headache, muscle aches, and nausea. He also experienced sweating on the fifth day of the cruise, towards the beginning of his symptom onset. Other possible symptoms for Legionella infection that were not experienced by Tom but are possible include malaise, lack of appetite (anorexia), lethargy, vomiting, and diarrhea (4, 5).

Pneumonia is a possible symptom as well, but only in Legionnaires’ disease (1). Patients with Pontiac fever will not experience pneumonia (1).

Key HPI Elements

  1. Location: The location of the pain is not explicitly mentioned, however, Tom did experience headaches and muscle aches, which suggest that his head and muscles are in pain. Additionally, his lungs are affected as he was diagnosed with pneumonia.
  2. Quality: The quality of Tom’s symptoms are not specified in this case.
  3. Quantity or severity: The severity of Tom’s symptoms are not specified in this case.
  4. Timing: Tom’s first symptoms occurred on the fifth day of his cruise. His symptom onset timing very likely happened within the typical incubation period of 2-14 days for Legionellosis (5). The start of his symptoms were when he woke up in a sweat at night on the fifth day. His symptoms also likely persisted for the remaining two days of the cruise.
  5. Setting: Tom very likely got infected on the cruise ship, and specifically because of his consistent and high use of the hot tubs on the ship, as Legionella infection commonly occurs through the aerosolization of particles from water systems (6).
  6. Factors: Tom has a history of asthma and experienced a recent flare-up. He used corticosteroids to deal with the flare-up, which may contribute to his risk factor due to their immunosuppressive effects (7). He is also over the age of 50 and male, which are additional risk factors for Legionella infection (6).
  7. Association manifestations: Pneumonia, but because it is assumed that Tom has Legionnaires’ disease and not Pontiac fever, there are no other associated manifestations that have been indicated.

References

  1. Gerber JS. 2020. Chapter 235, Legionella. In Kliegman et al. (ed), Nelson Textbook of Pediatrics. Elsevier. https://www-clinicalkey-com.ezproxy.library.ubc.ca/#!/content/book/3-s2.0-B9780323529501002352
  2. Fields BS, Haupt T, Davis JP, Arduino MJ, Miller PH, Butler JC. Pontiac fever due to Legionella micdadei from a whirlpool spa: possible role of bacterial endotoxin. J Infect Dis. 2001 Nov 15;184(10):1289-92. doi: 10.1086/324211.
  3. Brady MF, Sundareshan V. 2022. Legionnaires' Disease. In StatPearls [Internet]. StatPearls Publishing. https://pubmed.ncbi.nlm.nih.gov/28613558/
  4. Mohapatra RK, Mahal A, Kandi V, Kutikuppala LVS, Sarangi AK, Mishra S. 2022. Emerging pneumonia-like illness “legionellosis” in Argentina in the COVID-19 era: Cause to panic? Front. Pharmocol. 13. https://doi.org/10.3389/fphar.2022.1063237
  5. Cunha BA, Burillo A, Bouza E. 2016. Legionnaires' disease. Lancet 387(10016): 376-385. https://doi.org/10.1016/S0140-6736(15)60078-2
  6. Elsevier Point of Care. 2022. Legionnaires' Disease. https://www.clinicalkey.com/#!/content/clinical_overview/67-s2.0-9b6a05f2-3f43-445f-83ba-fed27dc8ba0f
  7. Youssef J, Novosad SA, Winthrop KL. Infection risk and safety of corticosteroid use. Rheumatic Disease Clinics. 2016 Feb 1;42(1):157-76.


(ii) Which body systems are affected? In what way has the normal physiological functioning of this body system been disturbed by the infection (specifically looking at the physiological changes without detailing the bacterial mechanism of this disturbance as that is the basis of another question). Representing this diagrammatically is helpful to demonstrate understanding.

Effects on the Respiratory System: Primarily, the L. pneumophila disrupts normal physiological functioning of the respiratory system (the lower respiratory tract) which results in pneumonia. The trachea is the initial infection and replication site of Legionella. (1). Then, the bacterium spreads to the lower respiratory tract and invades alveolar epithelial cells (1).

Once the pathogen enters the lungs, TLR-mediated recognition by innate immune cells including alveolar macrophages and dendritic cells initiates an inflammatory response (2). Pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and chemokines are released, which recruits more immune cells to the site of infection. Immune infiltration can cause tissue damage and impair lung function (3,4,5,6). As a result, blood vessels become highly permeable, thereby increasing serum osmolality and fibrin deposition in the alveoli (7).

Intracellular survival in macrophages allows the pathogen to spread to other organs (2). This can further exacerbate the inflammatory response while contributing to the infection’s systemic spread in the human host. In macrophages, the pathogen can interfere with antigen presentation and inhibit apoptosis, thereby prolonging its survival in infected cells and evading immune recognition (8,9). L. pneumophila modulates phagosome transport by modifying the vacuolar membrane into an endoplasmic reticulum-like replicative vacuole. Bacterial effectors then suppress the unfolded protein response which, in extreme cases, would have resulted in apoptosis (8,9,10,11).

The pathogen’s virulence factors cause direct damage to respiratory epithelium resulting in increased permeability and fluid leakage into alveolar spaces. This can contribute to the development of pulmonary edema, a common complication associated with Legionnaires' disease (12,13). The accumulation of fluid and cellular debris resulting from the immune response in the alveolar spaces, impairs gas exchange and reduces O2 uptake, leading to hypoxia and respiratory failure. In severe cases, this can cause life-threatening acute respiratory distress syndrome (14,15).

Effects on the Circulatory System & the Central Nervous System: The primary infection site of Legionella spp. is the respiratory tract (7). However, disseminated infection to other sites is possible through invading macrophages and entering the bloodstream (7). Possible extrapulmonary manifestations include myocarditis, encephalomyelitis and more (7).


References

  1. Winn WC. Legionella. Manual of clinical microbiology. 1995;7:572-85.
  2. Tan LT, Tee WY, Khan TM, Ming LC, Letchumanan V. Legionella pneumophila—The causative agent of Legionnaires’ disease. Progress In Microbes & Molecular Biology. 2021 Apr 8;4(1).
  3. Shin S, Case CL, Archer KA, Nogueira CV, Kobayashi KS, Flavell RA, Roy CR, Zamboni DS. Type IV secretion-dependent activation of host MAP kinases induces an increased proinflammatory cytokine response to Legionella pneumophila. PLoS pathogens. 2008 Nov 28;4(11):e1000220.
  4. Chauhan D, Shames SR. Pathogenicity and Virulence of Legionella: Intracellular replication and host response. Virulence. 2021 Dec 31;12(1):1122-44.
  5. Friedman H, Yamamoto Y, Klein TW. Legionella pneumophila pathogenesis and immunity. In Seminars in pediatric infectious diseases 2002 Oct 1 (Vol. 13, No. 4, pp. 273-279). WB Saunders.
  6. Kimizuka Y, Kimura S, Saga T, Ishii M, Hasegawa N, Betsuyaku T, Iwakura Y, Tateda K, Yamaguchi K. Roles of interleukin-17 in an experimental Legionella pneumophila pneumonia model. Infection and immunity. 2012 Mar;80(3):1121-7.
  7. Trousil J, Frgelecová L, Kubíčková P, Řeháková K, Drašar V, Matějková J, Štěpánek P, Pavliš O. Acute Pneumonia Caused by Clinically Isolated Legionella pneumophila Sg 1, ST 62: Host Responses and Pathologies in Mice. Microorganisms. 2022 Jan 14;10(1):179. doi: 10.3390/microorganisms10010179.
  8. Banga S, Gao P, Shen X, Fiscus V, Zong WX, Chen L, Luo ZQ. Legionella pneumophila inhibits macrophage apoptosis by targeting pro-death members of the Bcl2 protein family. Proceedings of the National Academy of Sciences. 2007 Mar 20;104(12):5121-6.
  9. Gao LY, Abu Kwaik Y. Activation of caspase 3 during Legionella pneumophila-induced apoptosis. Infection and immunity. 1999 Sep 1;67(9):4886-94.
  10. Roy CR, Tilney LG. The road less traveled: transport of Legionella to the endoplasmic reticulum. The Journal of cell biology. 2002 Aug 8;158(3):415.
  11. Treacy-Abarca S, Mukherjee S. Legionella suppresses the host unfolded protein response via multiple mechanisms. Nature communications. 2015 Jul 29;6(1):7887.
  12. Baskerville A, Conlan JW, Ashworth LA, Dowsett AB. Pulmonary damage caused by a protease from Legionella pneumophila. British journal of experimental pathology. 1986 Aug;67(4):527.
  13. Jäger J, Marwitz S, Tiefenau J, Rasch J, Shevchuk O, Kugler C, Goldmann T, Steinert M. Human lung tissue explants reveal novel interactions during Legionella pneumophila infections. Infection and immunity. 2014 Jan;82(1):275-85.
  14. Narita Y, Naoki K, Horiuchi N, Hida N, Okamoto H, Kunikane H, Watanabe K. A case of legionella pneumonia associated with acute respiratory distress syndrome (ARDS) and acute renal failure treated with methylprednisolone and sivelestat. Nihon Kokyuki Gakkai Zasshi= the Journal of the Japanese Respiratory Society. 2007 May 1;45(5):413-8.
  15. Gorman D, Green A, Puri N, Dellinger P. Severe ARDS secondary to Legionella pneumonia requiring VV ECMO in the setting of newly diagnosed hairy cell leukemia. Journal of Investigative Medicine High Impact Case Reports. 2022 Jan;10:23247096211065618.

(iii) What antibiotics/treatments might have been given (i.e., what are antibacterial treatments and what is their mechanism of action; are their any beneficial effects outside of direct antibacterial action? Representing this diagrammatically is helpful to demonstrate understanding.

The doctor on the cruise prescribed azithromycin to Tom on day 5 of the trip. Antibiotics with different mechanisms of actions and structures are being used in pneumonia depending on the causative pathogen and severity of disease (1, 2, 3). The first line agents for Legionella spp. treatments are respiratory quinolones like levofloxacin or  macrolides like azithromycin, and the second line option is doxycycline (1, 2, 3, 4).

Mechanism of Action:

1) Quinolones: levofloxacin and moxifloxacin belong to respiratory quinolones which are active against Legionella and kill (bactericidal property) the bacteria by blocking topoisomerase II and topoisomerase IV enzymes of bacteria (2, 3, 5). This inhibition will prevent the DNA synthesis of targeted bacteria (3, 5). Quinolone's monotherapy covers both typical and atypical bacterial pneumonia (6). As a result, if both typical and atypical characteristics were presented in the patient, respiratory quinolone is a proper 1st line agent because of the broad coverage (1, 6). Moreover, quinolone’s monotherapy is superior to macrolides and doxycycline when Legionella is diagnosed (1, 2, 3).

2) Azithromycin: this antibiotic is a broad spectrum macrolide which covers Legionella, Bartonella. henselae and Neisseria. gonorrhoeae (1, 7). It will inhibit the protein synthesis of bacteria by binding to the 23S rRNA of the 50s ribosomal subunits (8). This antibiotic will be transferred in higher concentration to the site of the infection due to the high accumulation in the phagocytes and high rate of distribution compared to other macrolides (9). Azithromycin is a suitable therapy in Legionella because of the reduction in the biofilm production which this bacteria is able to tolerate (9). Moreover, azithromycin is a basic antibiotic which makes it more effective in penetration of cell membranes and fighting against gram negative bacteria like Legionella (9). The regimen for oral route is 500 mg tablet on the first day, then 250 mg tablet per day for another 4 days or 500 mg tablet per day for 3 days (1).

3) Doxycycline: this antibiotic belongs to the tetracyclines (10). Doxycycline binds to the both 30S and 50S subunits of bacterial ribosomes and inhibits protein synthesis (10). Doxycycline is more effective in eliminating Legionella compared to erythromycin and tetracycline medications (6). The proper regimen for Legionella infection is doxycycline 200 mg tablet on the first day, then 100 mg tablet daily (1). However, in case of moderate-severe Legionella pneumonia, patients are recommended to take a full dose of 400 mg daily for 3 days, then 200 mg daily for the rest of the treatment (6).

References:

1) Loeb.Mark. Community-Acquired Pneumonia. In: e-CTC[Internet]. Ottawa (ON): Canadian Pharmacists Association. C2023[updated 2022, May 02; cited 2023 Mar 07]. Available from: http://www.myrxtx.ca. Also available in paper copy from the publisher.

2) Bennett JE, Dolin R, Blaser MJ. Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases. 9th Ed. Philadelphia, PA: Elsevier; 2020

3) Torres, Antoni et al. “Pneumonia.” Nature reviews. Disease primers vol. 7,1 25. 8 Apr. 2021, doi:10.1038/s41572-021-00259-0

4) Chahin, Abdullah, and Steven M Opal. “Severe Pneumonia Caused by Legionella pneumophila: Differential Diagnosis and Therapeutic Considerations.” Infectious disease clinics of North America vol. 31,1 (2017): 111-121. doi:10.1016/j.idc.2016.10.009

5) e-CPS [Internet]. Ottawa (ON): Canadian Pharmacists Associations, c2023[updated 2018, Mar 01; cited 2023 Mar 07]. Fluoroquinolones [CPhA monograph]. Available from: http://www.myrxtx.ca. Also available in paper copy from the publisher.

6) Cunha, B A. “The atypical pneumonias: clinical diagnosis and importance.” Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases vol. 12 Suppl 3 (2006): 12-24. doi:10.1111/j.1469-0691.2006.01393.x

7) Heidary M, Ebrahimi Samangani A, Kargari A, Kiani Nejad A, Yashmi I, Motahar M, Taki E, Khoshnood S. 2022. Mechanism of action, resistance, synergism, and clinical implications of azithromycin. Jo of Clinl Lab Anal 36:e24427.

8) e-CPS [Internet]. Ottawa (ON): Canadian Pharmacists Associations, c2023[updated 2019, Mar 05; cited 2023 Mar 07]. Azithromycin [CPhA monograph]. Available from: http://www.myrxtx.ca. Also available in paper copy from the publisher.

9) Parnham MJ, Haber VE, Giamarellos-Bourboulis EJ, Perletti G, Verleden GM, Vos R. 2014. Azithromycin: Mechanisms of action and their relevance for clinical applications. Pharmacology & Therapeutics 143:225–245

10) e-CPS [Internet]. Ottawa (ON): Canadian Pharmacists Associations, c2023[updated 2018, Jan 08; cited 2023 Mar 07]. Tetracyclines [CPhA monograph]. Available from: http://www.myrxtx.ca. Also available in paper copy from the publisher.

(iv) What is the significance of the exposure to hot tubs in this case - discuss Legionella lifecycle when answering this? Is Legionella a reportable communicable disease? What could be done to identify the source of these Legionella infections?

What is the significance of the exposure to hot tubs in this case?:

Hot tub exposure is significant to this case because it is the likely source of Tom’s infection. Just like regular swimming pools, hot tubs contain chlorine as well. However, Legionella is resistant to chlorine (1). Legionella thrives at temperatures between 25°C - 40°C (2), and hot tubs are set to 38°C - 40°C (3). The combination of chlorine resistance and ideal temperature makes hot tubs a great place for Legionella to inhabit.

Legionella Life Cycle:

Figure 1. Legionella life cycle (4)


Replication and transmission are the two main stages that Legionella undergoes (4). The full life cycle is as follows (4):

  • Amoeba takes up Legionella via phagocytosis
  • Replication occurs when nutrients are abundant in the amoeba
  • They prepare to be transmitted into the environment when nutrient levels are low
  • Achieve its mature intracellular form
  • And lysis of amoeba

Parasitized amoeba can also form biofilms, which are critical to the environmental survival and replication phases of Legionella’s life cycle (9). Biofilms are difficult to eliminate, granting Legionella additional protection against environmental conditions and sanitation methods, which provide it with conditions that are favorable for replication (10). This means that traits that promote transmission are repressed, and intracellular bacteria multiply (4). Once nutrients in the host cells deplete, Legionella switches to the transmissive phase, and starts to express genes to help it escape from the host cell and survive in the environment (4). If biofilm is found in a water system, like the pipes of the cruise’s hot tub system, pressure and vibration can cause pieces of it to dislodge, allowing it to colonize other parts of the water distribution system (9)

Figure 2. BC Center for Disease Control's Legionella outbreak protocol (10)

Is Legionella a reportable communicable disease?:

Yes, Legionella is a reportable communicable disease (11) under the BC Public Health Act (10). On the national level, public health officials must report every case within the first 30 days (12). For travel-associated cases, public health officials must report the case within 7 days to prevent infection from spreading to other nations or states (12). The List of Reportable Communicable Diseases in British Columbia is defined as a component of the Health Act Communicable Disease Regulations of B.C.’s Public Health Act. Legionellosis is listed under “Schedule B”, and as such is reportable only from laboratories to the Medical Health Officer (13). In Tom’s case, the cruise ship must undergo sterilization and identify the source of the infection to prevent new cases on the next voyage.

What could be done to identify the source of these Legionella infections?

This figure outlines the specific steps the BC Centre for Disease Control takes to investigate an outbreak of Legionella.

Health care teams in the regional health authorities arrange an interview time with the Legionella infection cases and write all important information in the “BCCDC case report form” (10). By doing this interview, health care teams will be able to recognize the infected sources (community, hospital, travel) and prevent further infection (10). The common source of pneumonia infection in this case was a cruise ship which the health care teams were required to gather information regarding the date and duration of travel, places with high risk bacterial exposure (Hot tub, shower, pool), and all the spaces that were being used during the trip (10). The hot tubs in the cruise ships need to be cleaned properly.

For single case identifications, obtaining the patient’s exposure history will help determine the source of the infection (10).

On the other hand, for outbreak investigations, a team must also be assembled before data is collected (10). Ideally, descriptive epidemiology in addition to environmental and microbiological data can be used to develop hypotheses for the environmental source of the outbreak (10). These hypotheses can then be tested and confirmed through site inspections and microbiological data (10). The CDC also suggests that comparing clinical and environmental samples through the use of serological or molecular techniques (ex. genome sequencing) can also help identify the source (14).

References:

  1. Mohapatra RK, Mahal A, Kandi V, Kutikuppala LVS, Sarangi AK, Mishra S. 2022. Emerging pneumonia-like illness “legionellosis” in Argentina in the COVID-19 era: Cause to panic? Frontiers in Pharmacology 13.
  2. Bennett JE, Dolin R, Blaser MJ. Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases. 9th Ed. Philadelphia, PA: Elsevier; 2020.
  3. 2020. Recommended Hot Tub Water Temperature | Canadian Home Leisure. Canadian Home Leisure. https://canadianhomeleisure.ca/recommended-hot-tub-water-temperature/. Retrieved 10 March 2023.
  4. Molofsky AB, Swanson MS. 2004. Differentiate to thrive: lessons from the Legionella pneumophila life cycle. Molecular Microbiology 53:29–40.
  5. Muder RR, Yu VL, Woo AH. 1986. Mode of Transmission of Legionella pneumophila: A Critical Review. Archives of Internal Medicine 146:1607–1612.
  6. Public Health Agency of Canada. 2013. Legionella. education and awareness. https://www.canada.ca/en/public-health/services/infectious-diseases/legionella.html. Retrieved 16 March 2023.
  7. Torres A, Cilloniz C, Niederman MS, Menéndez R, Chalmers JD, Wunderink RG, van der Poll T. 2021. Pneumonia. 1. Nat Rev Dis Primers 7:1–28.
  8. Gonçalves IG, Simões LC, Simões M. 2021. Legionella pneumophila. Trends in Microbiology 29:860–861.
  9. Abdel-Nour M, Duncan C, Low DE, Guyard C. 2013. Biofilms: The Stronghold of Legionella pneumophila. Int. J. Mol. Sci. 14(11): 21660-21675. doi: 10.3390/ijms141121660
  10. BC Center for Disease Control. 2021. Chapter I – Management of Specific Diseases Legionella outbreak investigation and control. Retrieved from http://www.bccdc.ca/resource-gallery/Documents/Guidelines%20and%20Forms/Guidelines%20and%20Manuals/Epid/CD%20Manual/Chapter%201%20-%20CDC/BC%20Legionella%20Guidelines%202021%20July%20FINAL.pdf.
  11. Centers for Disease Control and Prevention. 2022. Legionnaires Disease and Pontiac Fever: For Media. Retrieved from https://www.cdc.gov/legionella/qa-media.html.
  12. Centers for Disease Control and Prevention. 2021. How to Report Legionnaires Disease Cases. Retrieved from https://www.cdc.gov/legionella/health-depts/surv-reporting/report-cases.html
  13. Centers for Disease Control and Prevention. 2021 Controlling Legionella in Hot Tubs Retrieved from https://www.cdc.gov/legionella/wmp/control-toolkit/hot-tubs.html
  14. Centers for Disease Control and Prevention. 2021. Legionnaires Disease Diagnosis, Treatment, and Prevention. Retrieved from https://www.cdc.gov/legionella/clinicians/diagnostic-testing.html.


2. The Microbiology Laboratory

(i) What are the most common pathogens that cause pneumonia?

There are a variety of pathogens that can cause pneumonia depending on the age of the host. In neonates (up to 20 days in age), the following bacteria can cause pneumonia: Group B streptococcus, Escherichia coli , and Listeria monocytogenes. For those in the age range of 3 weeks to 3 months, there are bacterial and viral pathogens that can cause this illness. Bacterial pathogens include Chlamydia trachomatis and S. pneumoniae whereas viral pathogens include adenovirus, influenza, parainfluenza virus 1,2, and 3, along with respiratory syncytial virus. Those in the age range of 4 months to 5 years similarly can get infected by both bacterial and viral means. The bacterial pathogens include Chlamydia pneumoniae, Mycoplasma pneumoniae, S. pneumoniae and the viral pathogens include adenovirus, influenza, parainfluenza, rhinovirus, and respiratory syncytial virus. From age 5 to adolescence, the most common pathogens are bacterial and include C. pneumoniae, M. pneumoniae, and S. pneumoniae1. For those above the age of 18, the most common pathogens are M. pneumoniae, S. pneumoniae, C. pneumoniae, H. influenzae (type b,  nontypable), influenza and coronaviruses, and adenoviruses2. In immunocompromised patients, Haemophilus influenzae, Influenza A and B viruses, Streptococcus pneumoniae, Mycoplasma pneumonia are common along with other infections from other opportunistic pathogens3.


References

  1. Ostapchuk, M., Roberts, D. M., & Haddy, R. (2004, September 1). Community-acquired pneumonia in infants and children. American Family Physician. Retrieved March 10, 2023, from https://www.aafp.org/pubs/afp/issues/2004/0901/p899.html
  2. Marcdante, K., & Kliegman, R. M. (2018). Pneumonia. In Nelson Essentials of Pediatrics E-Book (pp.351-366). essay, Elsevier.
  3. Aleem, M., Sexton, R., & Akella, J. (2022, July 25). Pneumonia in an immunocompromised patient - statpearls - NCBI bookshelf. National Library of Medicine. Retrieved March 10, 2023, from https://www.ncbi.nlm.nih.gov/books/NBK557843/

(ii) How is pneumonia diagnosed, generally? What laboratory samples are taken and why? Is there other diagnostic testing that should be performed?

The most common means of diagnosis is a chest radiograph. Though it helps diagnose the extent and location of pneumonia, it cannot specify the causative germ4. Other methods include pulse oximetry to measure oxygen levels, sputum tests, and blood tests 1, 2,4. In older or severely ill patients, doctors may request a CT scan or pleural fluid culture4.

Figure 1. Legionella Diagnosis Methods

Several types of samples can be taken. For bacterial and mycobacterial cultures, blood sputum, pleural fluid, lung aspirates, urine, bronchoscopic specimens, and gastric aspirates are possible samples to be taken 3. Viral cultures may call for nasopharyngeal or oropharyngeal specimens, sputum, or lung aspirates. Culture samples provide good evidence of likely causative microorganisms since they are often taken directly from the site of infection 3. The aforementioned samples are useful for antigen detection and nucleic acid detection (PCR) tests 3. These are helpful  for detecting low levels of nucleic acid from multiple pathogens and do not depend on the viability of the target microbe 3. Antibody detection tests are for serum samples 3. Antibody detection has limited effects on diagnosis as it is rarely sufficient 3.


References

  1. NHLBI, “Pneuomonia Diagnosis,” Pneumonia Diagnosis, n.d.. [Online]. Available: https://www.nhlbi.nih.gov/health/pneumonia/diagnosis#:~:text=A%20chest%20X%2Dray%20is,enough%20oxygen%20into%20your%20blood. [Accessed: 10-Mar-2023].
  2. L. L. Hammitt, D. R. Murdoch, J. A. Scott, A. Driscoll, R. A. Karron, O. S. Levine, and K. L. O’Brien, “Specimen collection for the diagnosis of pediatric pneumonia,” Clinical Infectious Diseases, vol. 54, no. suppl_2, 2012.
  3. D. R. Murdoch, K. L. O’Brien, A. J. Driscoll, R. A. Karron, and N. Bhat, “Laboratory methods for determining pneumonia etiology in children,” Clinical Infectious Diseases, vol. 54, no. suppl_2, 2014.
  4. Mayo Clinic, “Pneumonia,” Mayo Clinic, 13-Jun-2020. [Online]. Available: https://www.mayoclinic.org/diseases-conditions/pneumonia/diagnosis-treatment/drc-20354210. [Accessed: 10-Mar-2023].

(iii) Explain the tests that can be performed specifically to diagnose Legionella infection.


The preferred diagnostic tests for Legionella infection are specimen culturing and urinary antigen test (UAT) (Table 1). Culturing remains the best method of diagnosis for Legionellosis because it can detect Legionella species and serogroups that cannot be done through the urinary antigen test. For culturing, the buffered charcoal yeast extract (BYCE) agar is the culture media specifically formulated for the isolation of Legionella spp.  (1,2,3) Sputum or endotracheal aspirates (commonly from suspected VAP patients) are already good specimens for culture, lung biopsy and bronchoscopy are usually not required. On the other hand, the UAT is the most common test done to diagnose Legionellosis. Around 97% of clinical diagnoses are obtained via the UAT as it can be easily performed even without special laboratory skills. (2,3) The test uses monoclonal antibodies to detect most Legionella pneumophila serogroup 1 lipopolysaccharide antigens but not other serogroups of L. pneumophila and other species  Legionella. L. pneumophila serogroup 1 causes 50%-80% of legionellosis so as many as 20%-50% of cases would not be detected if the urinary antigen test alone is used for diagnosis. Therefore, it is highly recommended to use culture and the UAT in combination. (1,2)

Other diagnostic tests for Legionella infection include direct fluorescence antigen (DFA), serology, and PCR. (2)

Table 1. Diagnostic testing characteristics for Legionnaires' disease

References:

  1. Centers for Disease Control and Prevention [Internet]. USA: NCIRD; 2021. Legionella (Legionnaires’ Disease and Pontiac Fever) Diagnosis, Treatment, and Prevention; 2021 [updated 2021 Mar 25; cited 2023 Mar 10]. Available from https://www.cdc.gov/legionella/clinicians/diagnostic-testing.html
  2. Pierre DM, Baron J, Yu VL, Stout JE. Diagnostic testing for Legionnaires' disease. Ann Clin Microbiol Antimicrob. 2017 Aug 29;16(1):59. Available from: https://doi: 10.1186/s12941-017-0229-6.
  3. Bennet JE, Dolin R, Blaser MJ. Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases. 9th Ed. Philadelphia, PA: Elsevier; 2020.

(iv) What are the expected results from the above tests? What are the test characteristics for these tests?

Legionella pneumophila antigen (Urine): Only detects L. pneumophila serogroup 1.1 Pharmaceutical companies manufacture kits to perform this test. A colour developer is added, and absorbance is read on a microplate reader. Samples with absorbances greater than three times the negative controls are considered positive.2

Serological tests: Acute and convalescent phase serum samples (3-10 weeks apart), however results do take significant time. Single titers are not helpful for diagnosis of Legionnaires. Cross reactivity among Legionella spp. and other bacteria are possible so the test is not very specific. Seroconversion may not be evident in a few cases.1

Legionella culture:

Examine all cultures after 72 to 96 hours of incubation. Streak the inoculated portion of each plate with a sterile loop to provide areas of heavy growth, and incubate the plates for 24 hours.3 Since Legionella is a relatively slow-growing bacteria, negative plates should be reincubated until 7 days post-inoculation and then re-examined for Legionella colonies.5

Direct fluorescent antibody (DFA): A positive slide has at least one brightly fluorescing apple green rod seen in the test slide (see Figure 2.) 5, with no fluorescing rods seen in the negative control. A sputum sample is the preferred specimen.5

Legionella species by PCR: A positive polymerase chain reaction (PCR) result for the presence of a specific sequence found within the Legionella 5S rRNA gene which indicates the presence of a Legionella species DNA.6

Figure 2. Direct Fluorescent Antibody

References

  1. Chaudhry, R., Sreenath, K., Agrawal, S. K., &Valavane, A. (2018). Legionella and legionnaires’ disease: Time to explore in India. Indian Journal of Medical Microbiology, 36(3), 324–333. https://doi.org/10.4103/ijmm.ijmm_18_298
  2. Dionne M, Hatchette T, Forward K. Clinical utility of a Legionella pneumophila urinary antigen test in a large university teaching hospital. Can J Infect Dis. 2003 Mar;14(2):85-8. doi: 10.1155/2003/642159. PMID: 18159429; PMCID: PMC2094914.
  3. Feeley, J.C., R.J. Gibson, G.W. Gorman, N.C. Langford, J.K. Rasheed, D.C. Mackel, and W.B. Baine. 1979. Charcoal-yeast extract agar: primary isolation medium for Legionella pneumophila. J Clin Microbiol. 10:437–441.
  4. Bridge JA, Edelstein PH. Oropharyngeal colonisation with Legionella pneumophila. J Clin Microbiol. 1983;18:1108–12.
  5. Haldane DJ, Peppard R, Sumarah RK. Direct immunofluorescence for the diagnosis of legionellosis. Can J Infect Dis. 1993 Mar;4(2):101-4. doi: 10.1155/1993/909761. PMID: 22346430; PMCID: PMC3250764.
  6. MacDonell MT, Colwell RR: The nucleotide sequence of the 5S rRNA from Legionella pneumophila. Nucleic Acids Res. 1987;15(3):1335. doi: 10.1093/nar/15.3.1335.

3. Bacterial Pathogenesis

(i) Encounter: where does the organism normally reside - geographically and host wise, and what are the bacterial characteristics that leave it suited to these places of residence.

Figure 1. Illustrates Legionella biofilm environment in the cross-section of the pipe [12].

Legionellosis is caused by Legionella bacteria. It strongly affects immunocompromised patients, causing pneumonia [1]. Most cases are caused by Legionella pneumophilia (L. pneumophilia), but L. bozemanae, L. micdadei, and L. longbeachae are other possible strains [2]. In total, Legionella spp. contain 58 species and 3 subspecies [3].

Legionella bacteria inhabit aquatic environments [4]. These environments may be natural or anthropogenic [4]. Natural environments include freshwater reservoirs such as lakes, rivers, groundwater, compost, and soil [4]; anthropogenic environments include water fountains, plumbing systems, bathtubs, air humidifiers, and hot tubs [4]. Legionella are gram-negative rods [4] but they exhibit strong pleomorphism and may become rod-shaped, coccoid, or filamentous in response to different environmental factors such as temperature, nutrient availability, habitat, and metabolite presence [4]. Legionella survives in temperatures between 0 and 68°C with the optimum growth temperature being around 35°C [4]; hot tub temperatures don’t exceed 40°C [5], supporting growth.

Host wise, humans can become infected with Legionella spp. when they inhale contaminated water droplets, such as those produced by a hot

Figure 2. Geographic distribution of cases in EU/EEA in 2019 [1]

tub or shower (6 ). The bacteria then enters the lungs and causes a range of respiratory symptoms, including fever, cough, shortness of breath, and pneumonia (7 ). In the lungs, Legionella spp. Almost exclusively infects alveolar macrophages and lives almost the entirety of its life cycle localized there (7). Because of its type 4 secretion system and over 300 effector proteins, which block phagosomal maturation and fusion with lysosomes, the bacteria does not have a need to keep moving around (7 ).

One of the key characteristics of Legionella spp. that suit it to these locations is its ability to produce multispecies biofilms, which enable high resistance to environmental factors [4]. Biofilms are complex interspecies microbiome structures that adhere to the surface, which the bacterium uses to its advantage [8, figure 1]. In biofilms, the Legionella quorum sensing (Lqs) system and transcription factor, LvbR, and temperature control phenotypic variations of the bacteria [9]. The twin-arginine translocation system (Tat) pathway [2], and the bffA gene [4] are important for biofilm formation [2, 4]. This biofilm-forming ability is a serious public health and ecosystem concern [4]. Biofilms enable long-term Legionella survival and enable pathogen persistence despite disinfection [4]. Biofilms have high resistance to antibiotics, are metabolically active, and express virulence genes, allowing them to be highly infectious and replicate within permissive hosts [8,9]. Water systems that contain nutrients, scale, and corrosion existing at warm temperatures with little water flow increase the chance of biofilm accumulation [3]. Surfaces that are acidic, hydrophilic, and negatively charged inhibit L. pneumophilia adhesion [4].

Legionella are intracellular parasites that typically infect freshwater protozoa but can also infect phagocytic monocytes and alveolar

Figure 3. Adaptation strategies of Legoniella [1]

macrophages [1, 4]. Infection of protozoa enables environmental resistance [4] and provides a protected environment for multiplication [10]. Legionella pneumophila sequesters into these hosts to survive in aquatic systems due to its hydrophobic cell surface, and its lipopolysaccharide (LPS) is much less toxic compared to other Gram-negative bacteria [11]. Since Legionella are intracellular, they must tolerate acidification, starvation, temperature changes, oxidative stress, and other host defense mechanisms [1]. Legionella must sometimes exist extracellularly; physiological, morphogenic, and metabolic changes facilitate intra/extracellular transitions during its multi-phasic life cycle [1]. Starvation and environmental stress induce Legionella to transition from a metabolically active, replicating form to a motile, stress-resistant, transmissible form [1]. Mature intracellular and viable but non-cultivable forms also occur [1]. Legionella requires L-cysteine, ferric ions, and a low pH (~6.9) to grow, and under aerobic conditions, 2-5 days are needed to produce colonies [11].

References

  1. Oliva G, Sahr T, Buchrieser C. The Life Cycle of L. pneumophila: Cellular Differentiation Is Linked to Virulence and Metabolism. Front Cell Infect Microbiol. 2018 Jan 19;8:3. doi: 10.3389/fcimb.2018.00003.
  2. Newton HJ, Ang DK, van Driel IR, Hartland EL. Molecular pathogenesis of infections caused by Legionella pneumophila. Clin Microbiol Rev. 2010 Apr;23(2):274-98. doi: 10.1128/CMR.00052-09.
  3. Cunha BA, Burillo A, Bouza E. Legionnaires' disease. Lancet. 2016;387(10016):376-385. doi:10.1016/S0140-6736(15)60078-2
  4. Kanarek P, Bogiel T, Breza-Boruta B. Legionellosis risk-an overview of Legionella spp. habitats in Europe. Environ Sci Pollut Res Int. 2022 Nov;29(51):76532-76542. doi: 10.1007/s11356-022-22950-9.
  5. CDC Centers for Disease Control and Prevention. Hot Tubs [Internet]. CDC Centers for Disease Control and Prevention; 2022 Apr 2. Available from: https://www.cdc.gov/healthywater/swimming/swimmers/hot-tub-user-information.html
  6. Girolamini L, Mazzotta M, Lizzadro J, et al. Sit bath systems: A new source of Legionella infection. PLoS One. 2020;15(11):e0241756.
  7. Ziltener P, Reinheckel T, Oxenius A. Neutrophil and Alveolar Macrophage-Mediated Innate Immune Control of Legionella pneumophila Lung Infection via TNF and ROS. PLoS Pathog. 2016;12(4):e1005591.
  8. Sharma D, Misba L, Khan AU. 2019. Antibiotics versus biofilm: An emerging battleground in Microbial Communities. Antimicrobial Resistance & Infection Control 8
  9. Chauhan D, Shames SR. 2021. Pathogenicity and virulence of legionella: Intracellular replication and host response. Virulence 12:1122–1144
  10. Lau HY, Ashbolt NJ. The role of biofilms and protozoa in Legionella pathogenesis: implications for drinking water. J Appl Microbiol. 2009 Aug;107(2):368-78. doi: 10.1111/j.1365-2672.2009.04208.x.
  11. Legionella and Coxiella. In: Ryan KJ. eds. Sherris & Ryan's Medical Microbiology, 8e. McGraw Hill; 2022. Accessed March 09, 2023. https://accessmedicine.mhmedical.com/content.aspx?bookid=3107&sectionid=260928232
  12. Centers for Disease Control and Prevention. 2018. Legionella growth and spread: For Healthcare Facilities. Centers for Disease Control and Prevention. Centers for Disease Control and Prevention. Accessed March 07, 2023 from https://www.cdc.gov/legionella/wmp/overview/growth-and-spread.html#:~:text=Legionella%2C%20the%20bacterium%20that%20causes,gets%20into%20building%20water%20systems.


(ii) Entry: what facilitates the entry of the bacteria into the human host? What are the molecular, cellular and/or physiological factors at play in the initial entry/adherence step from the point of view of the organism and the host? How would our patient have come in contact with this bacteria

Figure 1. Transmission routes & life cycle [9]

Legionella inhabits aquatic environments [1]. Human exposure to Legionella most frequently occurs during recreational activity [1]. The patient likely encountered Legionella in the hot tub, an anthropogenic environment that infects significant numbers of individuals when contaminated [1]. Entry into the patient most likely occurred by inhalation of contaminated bioaerosols from the water, but may also have occurred by consumption, or direct exposure to contaminated water [1].  Our patient’s compromised immune system due to his recent asthma flare-up and use of corticosteroid medication may have also made him more susceptible to infection. It is thought that the hydrophobic side chains of Legionella contribute to its ability to distribute aerosols [2]. Only one case of human-to-human transmission has been reported [1]. When inhaled as an aerosol, L. pneumophilia is engulfed by human alveolar macrophages and replicates in them [3].

At the alveoli, Legionella attaches to alveolar macrophages with help from their flagella, pili, and other proteins [2]. Penetration of the host

Figure 2. Depicts the Life Cycle of Legionella within eukaryotic host cells [7]

epithelial barrier is mediated by the bacterial peptidylprolyl cis-trans isomerase (PPIase), Mip [4]; Mip binds collagen and degrades the extracellular matrix [4]. Legionella entry is mediated by receptor-mediated phagocytosis or coiling phagocytosis [1, 5]. Legionella binds complement receptors on alveolar macrophages to trigger engulfment [6]. Phagocytosis of L. pneumophilia is also mediated by the host protein phosphatidylinositol 3 (PI-3)-kinase [4].

Additional host factors like the complement system components, CR1 and CR3, further contribute to entry by antibody-mediated neutralization and impairing phagocytosis [7]. Major outer membrane protein (MOMP) binds complement components C3 and C3bi and mediates bacteria uptake via the complement receptors CR1 and CR3 of macrophages (8). Interactions with CR1 and CR3, as well as Fc receptors, are important for the internalization of L. pneumophila as the neutralization of complement processes by antibodies prevents phagocytosis by host cells [7]. L. pneumophilia’s Dot/Icm type IV secretion system (T4SS) plays a role in bacterial entry [9,10] and proteins EnhC, LpnE, RtxA, LvhB2, and HtpB are also important for host cell invasion [4]. Through the use of a T4S system, which is encoded by 27 genes of the dot/icm gene cluster, effectors are injected into the host which then manipulate host cell processes such as membrane transport systems, inhibiting host cell apoptosis and modulating host cell signalling pathways (11, 12). Figure 3 shows a detailed summary of the Dot/icm effectors, what they bind to, and their various effects.  HtpB influences mitochondrial recruitment to L. pneumophilia containing vacuoles (LpCV) [4]. LpnE inhibits phagolysosomal fusion [4], to block LpCV acidification. RtxA is critical for bacterial entry by binding β2 integrins in the host cell [13].

Figure 3. Dot/icm effectors, what they bind to, and their various effects


References

  1. Kanarek P, Bogiel T, Breza-Boruta B. Legionellosis risk-an overview of Legionella spp. habitats in Europe. Environ Sci Pollut Res Int. 2022 Nov;29(51):76532-76542. doi: 10.1007/s11356-022-22950-9.
  2. Legionella and Coxiella. In: Ryan KJ. eds. Sherris & Ryan's Medical Microbiology, 8e. McGraw Hill; 2022. Accessed March 09, 2023. https://accessmedicine.mhmedical.com/content.aspx?bookid=3107&sectionid=260928232
  3. Oliva G, Sahr T, Buchrieser C. The Life Cycle of L. pneumophila: Cellular Differentiation Is Linked to Virulence and Metabolism. Front Cell Infect Microbiol. 2018 Jan 19;8:3. doi: 10.3389/fcimb.2018.00003.
  4. Newton HJ, Ang DK, van Driel IR, Hartland EL. Molecular pathogenesis of infections caused by Legionella pneumophila. Clin Microbiol Rev. 2010 Apr;23(2):274-98. doi: 10.1128/CMR.00052-09.
  5. Lau HY, Ashbolt NJ. The role of biofilms and protozoa in Legionella pathogenesis: implications for drinking water. J Appl Microbiol. 2009 Aug;107(2):368-78. doi: 10.1111/j.1365-2672.2009.04208.x.
  6. Winn WC Jr. Legionella. In: Baron S, editor. Medical Microbiology [Internet]. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996.
  7. Chauhan D, Shames SR. 2021. Pathogenicity and virulence of legionella: Intracellular replication and host response. Virulence 12:1122–1144.
  8. Yang Z, Chen Y, Zhang Q, Chen X, Deng Z. Major Outer Membrane Protein from Legionella pneumophila Inhibits Phagocytosis but Enhances Chemotaxis of RAW 264.7 Macrophages by Regulating the FOXO1/Coronin-1 Axis. Journal of immunology research. 2021;2021:9409777-11.
  9. Mascarenhas DP, Zamboni DS. Inflammasome biology taught by Legionella pneumophila. J Leukoc Biol. 2017 Apr;101(4):841-849. doi: 10.1189/jlb.3MR0916-380R.
  10. Krakauer T. Inflammasomes, Autophagy, and Cell Death: The Trinity of Innate Host Defense against Intracellular Bacteria. Mediators Inflamm. 2019 Jan 8;2019:2471215. doi: 10.1155/2019/2471215.
  11. Meir A, Macé K, Lukoyanova N, et al. Mechanism of effector capture and delivery by the type IV secretion system from Legionella pneumophila. Nat Commun. 2020;11(1):2864. Published 2020 Jun 8. doi:10.1038/s41467-020-16681-z
  12. Ge J, Shao F. Manipulation of host vesicular trafficking and innate immune defence by legionella Dot/Icm effectors. Cellular microbiology. 12/01/2011;13(12):1870-1880. doi: 10.1111/j.1462-5822.2011.01710.x.
  13. Yang J-L, Li D, Zhan X-Y. 2022. Concept about the virulence factor of legionella. Microorganisms 11:74.


(iii) Multiplication and Spread: does the organism remain extracellular or do they enter into cells? What are the cellular/physiological determinants of these events? Do the bacteria remain at the entry site or do they spread beyond the initial site? (e.g. are there secondary sites of infection) and, if they spread, why do the bacteria hone in on particular secondary sites.

Figure 1. L. pneumophilia morphological states during it’s life cycle [5]

LCV evades degradation by subverting host cell defensive pathways through effector mediation. After phagocytosis, Legionella virulence effectors regulate small GTPases to facilitate LCV biogenesis [1]. The LCV is essential for divulging endocytic trafficking and vacuole acidification [1].  Vacuole acidification occurs through vacuolar(v)-ATPase which pumps protons into the LCV [1]. Afterward, host vesicular trafficking is subverted as the LCV escapes the endocytic pathway by recruiting endoplasmic reticulum (ER) – derived vesicles, which are replaced by ribosomes [1, 2]. After bacterial engulfment, Dot/Icm T4SS blocks phagolysosomal fusion in LpCVs to instead form ribosome-studded phagosomes [3,4,5,6]. LpCV undergoes remodeling to resemble rough endoplasmic reticulum (RER) [5,7]; smooth vesicles rich in lipids and proteins such as Rab1, Rab2, and Rab6, and mitochondria are recruited to the LpCV [3,5,7]. Five minutes after entry, RER-derived vesicles are recruited by the Dot/Icm T4SS and fuse to LpCV with the help of Rab1, Rab2, and Rab6 [3, 6,7]. The LCV, studded with ribosomes and bacterial effectors, creates a replication permissive compartment [1,2]. Thus, Replication begins 4-10 hours after entry [7].

In cells, Legionella obtains nutrients by exploiting the endoplasmic reticulum for amino acids and mitochondria [4]. L. pneumophilia intracellular replication relies on host amino acid transporter solute carriers and the phagolysosomal transporter A (PhtA) [3]. Amino acids are a primary

Figure 2. Intracellular pathways targeted by T4SS [20]

carbon source for Legionella [4]. L. pneumophilia increases amino acid availability by ubiquitinating host proteins, inhibiting host translation, and modulating autophagy [3, 7]. Legionella produces other enzymes that aid in nutrient accumulation for growth including a chymotrypsin-like enzyme, caseinase, gelatinase, serum protein degrading protease, aminopeptidase, phosphatase, lipase, deoxyribonuclease, ribonuclease, cellulase, and starch hydrolysis enzymes [8]. The host cell ubiquitination system is usurped by Legionella to mediate intracellular replication [9]. Ubiquitination is a reversible post-translational modification, where ubiquitin molecules are attached to protein substrates for protein degradation [10]. Therefore, ubiquitination regulates the stability, functionality, and quantity of critical regulatory proteins [9]. Hence, 0Legionella encodes proteins with three-F-box domain proteins, which mimic ubiquitin ligases to catalyze protein degradation [9]. The three-F-box-containing proteins function in tandem with eukaryotic-like ankyrin proteins to modulate the host cell into a suitable proliferation niche [10,11].

After intracellular replication in LpCV, Legionella induces cytotoxicity and host cell lysis [5] in response to nutrient depletion. Morphologic and metabolic changes switch the bacteria from a replicative to a transmissive form [3]. Replicating L. pneumophilia are rod-shaped, slender, non-motile, have a wavy cell wall, and don’t express motility or cytotoxicity-related genes [3]. Virulent L. pneumophilia are rod-shaped, have stubby ends containing poly-3-hydroxybutyrate (PHB), a smooth thick cell wall, and express transmissivity-related genes such as flagella that enable host cell egress and transmission [3, 4]. The transition between forms depends on levels of guanosine tetraphosphate, a second messenger that recruits sigma factors to enact new gene expression profiles [3]. This biphasic strategy makes L. pneumophilia replication and transmission mutually-exclusive [3]. Host cell lysis is mediated by the bacterial protein intracellular multiplication T (Icm-T) [5]. Icm-T is a component of the bacterial Dot/Icm T4SS [12], a system involved in host entry, blocking phagolysosomal fusion, recruitment of RER-derived vesicles, ubiquitination, inhibition of apoptosis, activation of the stress response, inhibition of host translation, and bacterial egress [6,7]. The Dot/Icm T4SS translocates 300 effector proteins into host cells to promote L. pneumophilia intracellular replication [3].

To escape the host cell, L. pneumophilia induces pore-formation in membranes and triggers cell lysis [7]. Pores are triggered by inflammasome activation in response to flagella detection [7]. Legionella secretion pathway (LSP), a type II secretion system (T2SS), secretes other factors important for motility and cell membrane breakdown [7]. Mip, a bacterial PPIase enables dissemination by degrading extracellular matrices to promote epithelial barrier penetration [7]. Studies indicate that Legionella can penetrate the lung’s alveolar epithelial barrier, the pulmonary vessels, and surrounding endothelial cells [13,14, figure 3]. While pneumonia is the primary clinical manifestation of L. pneumophila, extrapulmonary manifestations have been documented [6]. Secondary infection sites include the spleen, kidney, liver, skeletal muscle, heart, skin, soft tissue, and brain; in the form of neurologic changes, rhabdomyolysis, acute renal failure, electrolyte abnormalities, and gastrointestinal sites of infection [16,18,19]. The exact mechanism of this is unknown, however, it is theorized that the bacteria spread through direct invasion into the muscle or release of endotoxin into circulation that causes muscle injury (20). It is also likely that this bacteria can spread to other part of the body due to its ability to invade host immune cells, cells that usually travels extensively throughout the body [13,14, 17].


References

  1. Chauhan D, Shames SR. 2021. Pathogenicity and virulence of legionella: Intracellular replication and host response. Virulence 12:1122–1144.
  2. Isberg RR, O'Connor TJ, Heidtman M. 2008. The legionella pneumophila replication vacuole: Making a cosy niche inside host cells. Nature Reviews Microbiology 7:13–24.
  3. Oliva G, Sahr T, Buchrieser C. The Life Cycle of L. pneumophila: Cellular Differentiation Is Linked to Virulence and Metabolism. Front Cell Infect Microbiol. 2018 Jan 19;8:3. doi: 10.3389/fcimb.2018.00003.
  4. 0Kanarek P, Bogiel T, Breza-Boruta B. Legionellosis risk-an overview of Legionella spp. habitats in Europe. Environ Sci Pollut Res Int. 2022 Nov;29(51):76532-76542. doi: 10.1007/s11356-022-22950-9.
  5. Lau HY, Ashbolt NJ. The role of biofilms and protozoa in Legionella pathogenesis: implications for drinking water. J Appl Microbiol. 2009 Aug;107(2):368-78. doi: 10.1111/j.1365-2672.2009.04208.x.
  6. Krakauer T. Inflammasomes, Autophagy, and Cell Death: The Trinity of Innate Host Defense against Intracellular Bacteria. Mediators Inflamm. 2019 Jan 8;2019:2471215. doi: 10.1155/2019/2471215.
  7. Newton HJ, Ang DK, van Driel IR, Hartland EL. Molecular pathogenesis of infections caused by Legionella pneumophila. Clin Microbiol Rev. 2010 Apr;23(2):274-98. doi: 10.1128/CMR.00052-09.
  8. White RC, Cianciotto NP. Assessing the impact, genomics and evolution of type II secretion across a large, medically important genus: the Legionella type II secretion paradigm. Microb Genom. 2019;5(6):e000273. doi:10.1099/mgen.0.000273
  9. Khweek AA, Amer A. 2010. Replication of legionella pneumophila in human cells: Why are we susceptible? Frontiers in Microbiology 1.
  10. Suresh B, Lee J, Kim K-S, Ramakrishna S. 2016. The importance of ubiquitination and Deubiquitination in cellular reprogramming. Stem Cells International 2016:1–14.
  11. Al-Khodor S, Price CT, Habyarimana F, Kalia A, Abu Kwaik Y. 2008. A Dot/ICM-translocated ankyrin protein of legionella pneumophila is required for intracellular proliferation within human macrophages and protozoa. Molecular Microbiology.
  12. Durie CL, Sheedlo MJ, Chung JM, Byrne BG, Su M, Knight T, Swanson M, Lacy DB, Ohi MD. Structural analysis of the Legionella pneumophila Dot/Icm type IV secretion system core complex. Elife. 2020 Sep 2;9:e59530. doi: 10.7554/eLife.59530.
  13. Iliadi V, Staykova J, Iliadis S, Konstantinidou I, Sivykh P, Romanidou G, Vardikov DF, Cassimos D, Konstantinidis TG. 2022. Legionella pneumophila: The journey from the environment to the blood. Journal of Clinical Medicine 11:6126.
  14. Wagner C, Khan AS, Kamphausen T, Schmausser B, Ünal C, Lorenz U, Fischer G, Hacker J, Steinert M. 2007. Collagen binding protein MIP enables legionella pneumophila to transmigrate through a barrier of NCI-H292 lung epithelial cells and extracellular matrix. Cellular Microbiology 9:450–462
  15. Prichard W, Fick L. When Diarrhea Can Become Deadly: Legionnaires' Disease Complicated by Bowel Obstruction. Case Rep Gastroenterol. 2016;10(3):781-786. Published 2016 Dec 20. doi:10.1159/000453657
  16. Winn WC Jr. Legionella. In: Baron S, editor. Medical Microbiology [Internet]. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 40. Available from: https://www.ncbi.nlm.nih.gov/books/NBK7619/
  17. Delicata M, Banerjee A. A rare presentation of Legionnaires' disease. BMJ Case Rep. 2015 Jul 1;2015:bcr2013201337. doi: 10.1136/bcr-2013-201337.
  18. Chitasombat MN, Ratchatanawin N, Visessiri Y. Disseminated extrapulmonary Legionella pneumophila infection presenting with panniculitis: case report and literature review. BMC Infect Dis. 2018 Sep 17;18(1):467. doi: 10.1186/s12879-018-3378-0.
  19. Khan A, Borum M. American Journal of Gastroenterology. 2018; 113 S1242.
  20. Mascarenhas DP, Zamboni DS. Inflammasome biology taught by Legionella pneumophila. J Leukoc Biol. 2017 Apr;101(4):841-849. doi: 10.1189/jlb.3MR0916-380R


(iv) Bacterial Damage: do the bacteria cause any direct damage to the host (vs. the damage fully attributable to the host response) and, what is the nature of the bacterial damage?

Alveolar and tissue destruction in Legionellosis is due to neutrophil and monocyte-mediated damage and bacterial enzymes [1]. The infection triggers the production of an inflammatory infiltrate of neutrophils and macrophages to the site of infection, cell necrosis, abscess formation, and small blood vessel inflammation [2]. The inflammation can cause damage to the lung tissue, leading to symptoms such as cough, fever, and chest pain. Detection of L. pneumophilia by toll-like receptors (TLRs) induces reactive oxygen species (ROS), type I interferon (IFN), NFκB activation of pro-inflammatory cytokines, and the mitogen-activated protein kinase (MAPK) pathway [3]. These inflammatory mediators and cells produce tissue damage in the lungs which destroys alveolar airspaces and severely compromises gas exchange and respiratory function [1].

Furthermore, L. pneumophilia flagella activate host inflammasomes [4], causing inflammation and cell death through caspase proteins [3,5]. Inflammasome-induced cell death occurs by pyroptosis, an inflammatory form of death that consists of pore-formation in the cell membrane, swelling, osmotic lysis, and the release of inflammatory cytokines: interleukins (IL) IL-1β and IL-18 [5]. IL-1β recruits neutrophils and produces fever [1,3]. IL-18 activates phagocytes [3]. Pyroptosis induces phagocytosis by neutrophils and ROS production [5], damaging nearby cells.

Direct damage by the bacteria is largely protease-mediated [6]. L. pneumophilia expresses a tissue-destructive protease that mediates most of the direct lung damage in infection [1, 6]. L. pneumophila produces a zinc metalloprotease, ProA, that directly damages the lung tissue [7,8]. It is thought that ProA aids in iron accumulation for the growth of Legionella through the breakdown of host transferrin [9]. Direct damage to the host by Legionella also occurs through the production of secondary lung abscesses [10]. These lung abscesses are necrosis and cavitation of the lung tissue and generally occur in immunocompromised individuals, who are at the highest risk for acquiring L. pneumophila infection [7,10]. In severe cases, Legionella spp. can cause acute respiratory distress syndrome (ARDS), a life-threatening condition that requires mechanical ventilation (11). Diffuse alveolar hemorrhage (DAH) is another side effect of ARDS in which ARDS leads to direct leakage of red blood cells into alveolar spaces, which can be fatal (11).

References

  1. Winn WC Jr. Legionella. In: Baron S, editor. Medical Microbiology [Internet]. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 40. Available from: https://www.ncbi.nlm.nih.gov/books/NBK7619/
  2. Lau HY, Ashbolt NJ. The role of biofilms and protozoa in Legionella pathogenesis: implications for drinking water. J Appl Microbiol. 2009 Aug;107(2):368-78. doi: 10.1111/j.1365-2672.2009.04208.x.
  3. Krakauer T. Inflammasomes, Autophagy, and Cell Death: The Trinity of Innate Host Defense against Intracellular Bacteria. Mediators Inflamm. 2019 Jan 8;2019:2471215. doi: 10.1155/2019/2471215.
  4. Newton HJ, Ang DK, van Driel IR, Hartland EL. Molecular pathogenesis of infections caused by Legionella pneumophila. Clin Microbiol Rev. 2010 Apr;23(2):274-98. doi: 10.1128/CMR.00052-09.
  5. Mascarenhas DP, Zamboni DS. Inflammasome biology taught by Legionella pneumophila. J Leukoc Biol. 2017 Apr;101(4):841-849. doi: 10.1189/jlb.3MR0916-380R.
  6. Baskerville A, Conlan JW, Ashworth LA, Dowsett AB. Pulmonary damage caused by a protease from Legionella pneumophila. Br J Exp Pathol. 1986 Aug;67(4):527-36.
  7. Chauhan D, Shames SR. Pathogenicity and Virulence of Legionella: Intracellular replication and host response. Virulence. 2021;12(1):1122-1144. doi:10.1080/21505594.2021.1903199
  8. Scheithauer L, Thiem S, Schmelz S, et al. Zinc metalloprotease ProA of Legionella pneumophila increases alveolar septal thickness in human lung tissue explants by collagen IV degradation. Cell Microbiol. 2021;23(5):e13313. doi:10.1111/cmi.13313
  9. White RC, Cianciotto NP. Assessing the impact, genomics and evolution of type II secretion across a large, medically important genus: the Legionella type II secretion paradigm. Microb Genom. 2019;5(6):e000273. doi:10.1099/mgen.0.000273
  10. Baron RM, Baron BW, Barshak M. Lung Abscess. In: Loscalzo J, Fauci A, Kasper D, Hauser S, Longo D, Jameson J. eds. Harrison's Principles of Internal Medicine, 21e. McGraw Hill; 2022. Accessed March 10, 2023. https://accessmedicine-mhmedical-com.ezproxy.library.ubc.ca/content.aspx?bookid=3095&sectionid=265415202
  11. Kashif M, Patel R, Bajantri B, Diaz-Fuentes G. Legionella pneumonia associated with severe acute respiratory distress syndrome and diffuse alveolar hemorrhage - A rare association. Respir Med Case Rep. 2017;21:7-11. Published 2017 Mar 14. doi:10.1016/j.rmcr.2017.03.008


4. The Immune Response

(i) Host response: what elements of the a.) innate and b.) adaptive (humoral and cellular) immune responses are involved in this infection? Describe the immune cell populations involved as well as key chemical mediators.

A complex set of innate immune responses are triggered when pathogen-associated molecular patterns (PAMPs) are recognized by pattern recognition receptors (PRPs) (1, 2). Molecular patterns associated with Legionella pneumophila (L. pneumophila) include lipopolysaccharide (LPS), heat shock proteins (HSPs), outer membrane proteins (OMPs), peptidoglycan, lipopeptides, flagella, and nucleic acids (1, 2, 3). PRPs include toll-like receptors (TLRs), nod-like receptors (NLRs), and RIG-I-like receptors (RLRs), where RIG stands for retinoic acid inducible gene I (1, 2, 3). The initial response to a Legionella infection typically consists of an interstitial inflammatory reaction involving macrophages, B lymphocytes, natural killer (NK) cells, and immature dendritic cells, which likely represent a first line of defense against the bacterium (2, 3).

As shown in Figure 1, once L. pneumophila is recognized by the PRPs, cell-autonomous mechanisms in the phagocytes are activated to limit the growth of the pathogen. Cytokines including interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α) and interleukins (IL) such as IL-6 and IL-1, and chemokines including macrophage inflammatory protein 1 β (MIP-1 β), monocyte chemotactic protein 2 (MCP-2) are produced by macrophages and lymphocytes including NK cells (2).

Both IFN-γ and TNF-α inhibit the growth of L. pneumophila (3). In addition, NK cells also kill macrophage targets that are infected by L. pneumophila (4). Other cytokines, e.g. IL-12, that are produced by macrophages and dendritic cells after Legionella infection induce Type 1 T helper (Th1) cells which are involved in adaptive immune responses. As for chemokines, their function is to stimulate the migration of cells, especially leukocytes, to the inflammatory sites (2).

Adaptive immunity is much simpler compared to innate immunity (4). Activated T cells, Th1 helper cytokine IFN-γ, and activated macrophages are involved in the restriction of intracellular growth and proliferation of L. pneumophila. Meanwhile, the role of antibodies in the immunopathogenesis of L. pneumophila is unclear and perhaps only as a second line of defense. In fact, instead of helping to eradicate L. pneumophila, antibodies to the bacterium have been shown to promote its uptake and replication in macrophages (2, 4).

Figure 1: Innate immune responses of a mammalian phagocyte (a monocyte, macrophage, or neutrophil) to Legionella pneumophila infection (2)

The severity of Legionella infection ranges from mostly asymptomatic to mild, as in the case of Pontiac fever, to severe pneumonia. In an experiment conducted on A/J mice, the mice developed an inflammatory immune response involving macrophages, B lymphocytes, and NK cells, etc after intratracheal inoculation with L. pneumophila (5). Mice that were immunocompetent, i.e. with no depletion or functional impairment of cluster of differentiation 4+ (CD4+) T cells and CD8+ T cells, were able to recover within a week. On the other hand, lethality increased in mice with depletion of both CD4+ and CD8+ T cells. In immunocompetent humans, Legionella infection can also be controlled adequately by the rapid innate immune response in 5-7 days (6).

Immunocompromised patients such as patients infected with the human immunodeficiency virus (HIV), patients suffering chronic pulmonary diseases and solid organ transplant patients who are receiving immunosuppressant treatment, and splenectomized patients, etc are more vulnerable to Legionella infection (7, 8, 9). People who are infected with HIV suffer a deficiency of CD4+ T cells and functional deficits in CD4+ T cells, CD8+ T cells, and NK cells (10).  Immunosuppression treatment weakens the patient’s immunity and increases the risk of the patient getting L. pneumophila.

References

1. Yamamoto Y, Klein TW, Newton C, Friedman H. Chapter 21: Innate and Adaptive Immunity to Legionella pneumophila. In: Marre R, Kwaik YA, Bartlett C, Cianciotto NP, Fields BS, Frosch M, Hacker J, Lück PC (eds), Legionella. 2001. https://doi.org/10.1128/9781555817985.ch21

2. Liu X, Shin S. 2019. Viewing Legionella pneumophila Pathogenesis through an Immunological Lens. Journal of Molecular Biology, Volume 431, Issue 21, p.4321-4344. https://doi.org/10.1016/j.jmb.2019.07.028.

3. Massis LM, Zamboni DS. Innate immunity to legionella pneumophila. Front Microbiol. 2011;2:109. doi:10.3389/fmicb.2011.00109

4. Yamamoto Y, Klein TW, Newton C, Friedman H. Chapter 21: Innate and Adaptive Immunity to Legionella pneumophila. In: Marre R, Kwaik YA, Bartlett C, Cianciotto NP, Fields BS, Frosch M, Hacker J, Lück PC (eds), Legionella. 2001. https://doi.org/10.1128/9781555817985.ch21

5. Susa M, Ticac B, Rukavina T, Doric M, Marre R. Legionella pneumophila Infection in Intratracheally Inoculated T Cell-Depleted or -Nondepleted A/J Mice. J Immunol 1 January 1998; 160 (1): 316–321. https://doi.org/10.4049/jimmunol.160.1.316

6. Brown AS, Yang C, Hartland EL, van Driel IR. The regulation of acute immune responses to the bacterial lung pathogen Legionella pneumophila. J Leukoc Biol. 2017;101(4):875-886. doi:10.1189/jlb.4MR0816-340R

7. Kao AS, Myer S, Wickrama M, Ismail R, Hettiarachchi M. Multidisciplinary Management of Legionella Disease in Immunocompromised Patients. Cureus. 2021;13(11):e19214. Published 2021 Nov 2. doi:10.7759/cureus.19214

8. Kümpers P, Tiede A, Kirschner P, Girke J, Ganser A, Peest D. 2008. Legionnaires' disease in immunocompromised patients: a case report of Legionella longbeachae pneumonia and review of the literature. Journal of Medical Microbiology, Volume 57, Issue 3. https://doi.org/10.1099/jmm.0.47556-0

In the case of patients who had splenectomy, they have lost the main filter for blood-borne pathogens and antigens, and during bacterial infections, a regulator of lymphocyte traffic and a site where lymphocytes are primed (8, 9).

(ii) During a bacterial infection, excessive inflammation can result in immune-mediated pathology. What happens to the immune response in more mild vs more severe illness forms? Describe what cell types are involved. What cellular/physiological features of immunocompromised hosts make them more vulnerable to Legionella?

The innate immune system is the most important for clearing Legionella infection (1). Immunocompromised patients with defective macrophages and T lymphocytes are more susceptible to LD, likely due to challenges with macrophage activation by IFN-γ secreted from natural killer and Th1 cells (2). Meanwhile, patients with humoral immune deficiency did not exhibit an increased incidence of LD (2). Infected macrophages secrete various pro-inflammatory chemokines and cytokines to trigger an inflammatory response (1). Various microbial products in the macrophage cytosol contribute to the inflammatory response signalling (1). Mouse caspase 11 and human homologs caspase 4 and caspase 5 are activated after binding cytosolic Legionella lipopolysaccharide (3). Specifically, caspase is important for septic shock in mice (3). The mouse nucleotide-binding oligomerisation domain (NOD)- like recpetor (NLR) protein, NAIP5, responds to bacterial flagellin, which leads to the secretion of IL-1b and IL-18 (3). NOD1 and NOD2 detect peptidoglycan fragments to sustain the pro-inflammatory signalling pathways (3). Furthermore, MAVS and RIG-1 proteins recognise bacterial nucleic acid to trigger type I interferon production (1).

In a mouse model of LD, cytokines released by macrophages in more severe inflammatory response include keratinocyte-derived chemokine (KC), TNF-α, macrophage inflammatory protein-2 (MIP-2), IL-12, IL-18, and IFN-γ (4). Moreover, humans demonstrate increased TNF-α and IL-8 during a Legionella infection (5). Legionella heat shock protein was shown to induce the production of IL-1, IL-6, TNF-α, IFN-γ and IL-1 (6). TNF-α enhances the bactericidal activity of macrophages and oxidative bursts of neutrophils (1). Individuals receiving TNF-α antagonist treatment are reported to have a higher incidence of LD (7). IL-12 induces Th1 cells to secrete TNF-α for macrophage activation (6). However, excessive mobilisation of these cytokines can result in immune-mediated pathology and tissue damage (1). The specific cytokines released by immune cells depend on the pathogenicity of the Legionella strain (1). For instance, monocyte chemotactic protein 3 (MCP-3) is only expressed in virulent strains of L. pneumophila, while MIP-1α is stimulated by both virulent and avirulent L. pneumophila (6).

1. Bennett JE, Dolin R, Blaser MJ, Douglas RG, Mandell GL. Bartonella, Legionnaires’ Disease and Pontiac Fever. In: Mandell, Douglas, and Bennett's principles and practice of infectious diseases. Philadelphia: Elsevier; 2020. p. 2807–2817.

2. Rajasurya V, Surani S. Legionnaires disease in immunocompromised host. Hospital Acquired Infection and Legionnaires' Disease. 2020;

3. Shi J, Zhao Y, Wang Y, Gao W, Ding J, Li P, et al. Inflammatory caspases are innate immune receptors for intracellular LPS. Nature. 2014;514(7521):187–92.

4. Archer KA, Ader F, Kobayashi KS, Flavell RA, Roy CR. Cooperation between multiple microbial pattern recognition systems is important for host protection against the intracellular pathogen legionella pneumophila. Infection and Immunity. 2010;78(6):2477–87.

5. Menéndez R, Sahuquillo-Arce JM, Reyes S, Martínez R, Polverino E, Cillóniz C, et al. Cytokine activation patterns and biomarkers are influenced by microorganisms in community-acquired pneumonia. Chest. 2012;141(6):1537–45.

6. Friedman H, Yamamoto Y, Klein TW. Legionella pneumophila pathogenesis and immunity. Seminars in Pediatric Infectious Diseases. 2002;13(4):273–9.

7. Tubach F, Ravaud P, Salmon-Ceron D, Petitpain N, Brocq O, Grados F, et al. Emergence of legionella pneumophila pneumonia in patients receiving tumor necrosis factor- antagonists. Clinical Infectious Diseases. 2006;43(10).

(iii) Bacterial survival and immune response evasion: how does the bacteria attempt to evade the host immune response? Consider molecular and/or cellular mechanisms targeting immune responses.


The pathogen is able to survive in the host primarily due to its Dot/Icm T4SS as it mainly targets alveolar macrophages (1). Its effector proteins allow the pathogen to evade phagosome-lysosome fusion and instead establish an endoplasmic-reticulum-derived Legionella-containing vacuole (LCV) to facilitate its replication and evade the host’s immune responses (1). Specifically, DotA is crucial to the assembly and activity of the T4SS, VipD binds to the host’s Rab5 to prevent its downstream functions, blocking vacuolar maturation and acidification, and SidF interacts with proapoptotic members of the Bcl112 family, inhibiting their prodeath functions in macrophages (1,2).

The concentration of Legionella is amplified through repeated cycles of phagocytosis, intracellular replication, escape into the extracellular environment and re-phagocytosis by macrophages (3). Legionella alters between a transmissive and replicative phase (3). Legionella in the transmissive phase is very small and highly motile have increased virulence to infect cells (3). Intracellular replication is characterised by a non-motile replicative phase (3). When nutrients are depleted, Legionella converts back into a transmissive flagellated form (4). Pathogen-induced apoptosis and cellular necrosis release Legionella to infect other macrophages (5). Legionella enters the host lungs through inhalation, where the bacteria is phagocytosed by alveolar macrophages and resides in nascent phagosomes (3). Intracellular replication in macrophages protects Legionella from host immune responses (4). The Legionella type IVb secretion system, icm/dot, releases effector proteins from the vacuole to the cytoplasm (6). This modulates host vesicular transport by inhibiting the fusion of the phagosome to acidic lysosomes (6). The effector proteins, RalF, SidM, LepB, LidA and SidJ, recruit membrane from the endoplasmic reticulum to form a specialised Legionella-containing vacuole, which provides a niche for intracellular replication and immune evasion (6). Other effectors such as SdhA, SidF, SidP and LepB are crucial for the structural integrity and assembly of the specialised vacuole (7). Many type IV effector protein sequences resemble eukaryotic proteins (3).

Furthermore, opsonisation-dependent phagocytosis by C3 can attenuate oxidative burst, further promoting intracellular survival (8). Some Legionella stains have Cu-Zn superoxide dismutase that provides resistance to oxidative burst chemical species, such as hydrogen peroxide, superoxide anion and hydroxyl radicals (5).

In addition, another way that Legionella manages to survive and evade host immune responses is by the host cells inability of recognizing the bacterium’s lipopolysaccharide LPS using toll-like receptor 4 (TLR4) (9). Normally, bacterial LPS is detected by TLR4 on immune cells which ultimately ignites both innate and adaptive immune responses, but Legionella LPS has an unusually long lipid A portion that consists of fatty acids and is not able to interact with CD14+ (9). Because of the lack of recognition, the host immune response does not become activated and the bacterium is able to continue its lifecycle (9).

References:

  1. Martynova E, Rizvanov A, Urbanowicz RA, Khaiboullina S. 2022. Inflammasome contribution to the activation of th1, th2, and th17 immune responses. Frontiers in Microbiology 13.  
  2. Best AM, Abu Kwaik Y. 2018. Evasion of phagotrophic predation by protist hosts and innate immunity of metazoan hosts bylegionella pneumophila. Cellular Microbiology 21.
  3. Bennett JE, Dolin R, Blaser MJ, Douglas RG, Mandell GL. Bartonella, Legionnaires’ Disease and Pontiac Fever. In: Mandell, Douglas, and Bennett's principles and practice of infectious diseases. Philadelphia: Elsevier; 2020. p. 2807–2817.
  4. Kagan JC, Roy CR. Legionella phagosomes intercept vesicular traffic from endoplasmic reticulum exit sites. Nature Cell Biology. 2002;4(12):945–54.
  5. Gao L-Y, Abu Kwaik Y. Apoptosis in macrophages and alveolar epithelial cells during early stages of infection by legionella pneumophila and its role in cytopathogenicity. Infection and Immunity. 1999;67(2):862–70.
  6. Shohdy N, Efe JA, Emr SD, Shuman HA. Pathogen effector protein screening in yeast identifies legionella factors that interfere with membrane trafficking. Proceedings of the National Academy of Sciences. 2005;102(13):4866–71.
  7. Creasey EA, Isberg RR. The protein SDHA maintains the integrity of the legionella -containing vacuole. Proceedings of the National Academy of Sciences. 2012;109(9):3481–6.
  8. Payne NR, Horwitz MA. Phagocytosis of legionella pneumophila is mediated by human monocyte complement receptors. Journal of Experimental Medicine. 1987;166(5):1377–89.
  9. Liu, X., & Shin, S. (2019, October 4). Viewing legionella pneumophila pathogenesis through an immunological lens. Journal of molecular biology. Retrieved March 10, 2023, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9332137/

(iv) Outcome: a.) is the bacteria completely removed, or can chronic infection occur? b.) can asymptomatic infection occur? and c.) is there sterilizing immunity to future infections from this particular bacteria? Consider immunocompetent and immunocompromised hosts.

After 5 to 7 days of proper antibacterial therapy, the pathogen will be eradicated in the immunocompetent host. (1) Immunocompromised host may experience disseminated LD, and the pathogen disseminates via macrophage. (1) Immunocompromised hosts may take up to 14-21 days of therapy. (1) Although symptoms like cough may persist, chronic infection will not occur once treated. (1) Failure to treatment of LD should reconsider the diagnosis, possibility of coinfection or superinfection, or another disease that complicates LD. (1) Most immunocompetent hosts remain asymptomatic, or infection is self-limited after exposure to Lp. (2) Most likely, immunocompromised hosts will not be asymptomatic as their immune system cannot stop bacteria from thriving. Some possibility for sterilizing immunity of Lp was shown by using flagella of Lp as antigen, and it suggested the possibility of a vaccine. (3) It may be possible only for the immunocompetent host, not the immunocompromised host because it involves T cells. (3) However, there is no vaccine available currently. (1) Reinfection is uncommon; however, prior LD does not prevent reinfection. (1)  

References

  1. Bennett JE, Dolin R, Blaser MJ. Chapter 232. Legionnaires’ Disease and Pontiac Fever. In: Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases. Philadelphia, PA: Elsevier; 2020.
  2. Newton, Hayley J., et al. "Molecular pathogenesis of infections caused by Legionella pneumophila." Clinical microbiology reviews 23.2 (2010): 274-298. Available from: https://journals.asm.org/doi/full/10.1128/cmr.00052-09#sec-33
  3. Park, B., Park, G., Kim, J. et al. Innate immunity against Legionella pneumophila during pulmonary infections in mice. Arch. Pharm. Res. 40, 131–145 (2017). https://doi.org/10.1007/s12272-016-0859-9