Course:PATH4172017W2/Case 3

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Case 3

A Cruise Holiday

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 in tow 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, relatively 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 a pneumonia. She explains that her presumptive diagnosis is that of Legionnaires disease and leaves Tom’s wife with a sterile sample container to collect whatever fluid Tom might cough up for delivery to her. She explains that she can do a microscopic examination on the respiratory fluid which will help in the diagnosis.

In the meantime she starts Tom on erythromycin and lets the family know that she will check in on Tom regularly over the next few days to monitor his progress. More people are diagnosed with a similar pneumonia over the next two days, mostly in people who came aboard with a slightly compromised immune system, like in Tom’s case. The cruise ship alerts the hospital at their next port of call in case any of the patients worsen enough to require hospitalization. When they arrive at port blood samples are collected from all of the patients and delivered to the hospital laboratory for serology. The ship also takes extra time in port to allow for a full scale sterilization regime to be performed on all of the hot tubs. At this stage Tom is feeling well enough to continue on the cruise, although at a slower pace than when he first boarded.

The Body System Questions

  1. What are the signs (objective characteristics usually noted/detected by a healthcare professional) and symptoms (subjective characteristics experienced by the patient).
  2. Which body system is effected. In what way has the normal physiological functioning of this area of the body been disturbed by the infection (without going into detail on the cause of this disturbance as this will be dealt with in questions 3 and/or 4). Representing this diagrammatically is always helpful. What made Tom susceptible to this infection?
  3. Why did the doctor prescribe erythromycin and how does this antibiotic work to rid the body of the organism?
  4. Would this be considered an ‘outbreak’, is it reportable and to what official body. What role might the hot tub have played in this infectious scenario?

The Microbiology Laboratory Questions

  1. Other than the stated bacterial cause, what are the most common bacterial pathogens associated with this type of infectious scenario.
  2. What are all the samples that could be taken for laboratory testing (including the blood and ‘sputum’ in this case) and how important is the Microbiology Laboratory in the diagnosis of this particular infectious disease?
  3. Explain the tests that will be performed on the samples in order to detect any of the potential bacterial pathogens causing this disease.
  4. What are the results expected from these tests allowing for the identification of the bacteria named in this case.

Bacterial Pathogenesis Questions

Using the following pathogenic steps outline the pathogenesis of the bacteria named as being responsible for this infection.

  1. 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. How would our patient have come in contact with this bacteria
  2. 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.
  3. Multiplication and Spread: does the organism remain extracellular or do they enter into cells and what are the molecular and cellular determinants of these events. Do the bacteria remain at the entry site or do they spread beyond the initial site i.e. are there secondary sites of infection and why do the bacteria hone in on these particular secondary sites.
  4. Bacterial Damage: do the bacteria cause any direct damage to the host (or is the damage fully attributable to the host response, as indicated below) and, if so, what is the nature of the bacterial damage. Can it be linked to any of the signs and symptoms in this case

The Immune Response

  1. Host response: what elements of the innate and adaptive (humoral and cellular) immune response are involved in this infection.
  2. Host damage: what damage ensues to the host from the immune response?
  3. Bacterial evasion: how does the bacteria attempt to evade these host response elements.
  4. Outcome: is the bacteria completely removed, does the patient recover fully and is there immunity to future infections from this particular bacteria ?

Reports

A Cruise Holiday

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 in tow 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, relatively 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 a pneumonia. She explains that her presumptive diagnosis is that of Legionnaires disease and leaves Tom’s wife with a sterile sample container to collect whatever fluid Tom might cough up for delivery to her. She explains that she can do a microscopic examination on the respiratory fluid which will help in the diagnosis.

In the meantime she starts Tom on erythromycin and lets the family know that she will check in on Tom regularly over the next few days to monitor his progress. More people are diagnosed with a similar pneumonia over the next two days, mostly in people who came aboard with a slightly compromised immune system, like in Tom’s case. The cruise ship alerts the hospital at their next port of call in case any of the patients worsen enough to require hospitalization. When they arrive at port blood samples are collected from all of the patients and delivered to the hospital laboratory for serology. The ship also takes extra time in port to allow for a full scale sterilization regime to be performed on all of the hot tubs. At this stage Tom is feeling well enough to continue on the cruise, although at a slower pace than when he first boarded.

The Body Systems Questions

Question 1

What are the signs (objective characteristics usually noted/detected by a healthcare professional) and symptoms (subjective characteristics experienced by the patient).

Legionella is a water and soil organism, and Legionella pneumophila is a gram-negative non-enteric bacilli that loves aqueous and moist environments, known to cause a small percentage of community- and hospital-acquired pneumonia (Glover and Reed, 2008). It is transmitted by aerosolization and can produce localized outbreaks relating to excavation sites and contaminated water (Glover and Reed, 2008). Infection is rare in < 30 year old subjects, having competent immune systems, but additional risk is introduced by travel history, age, and chronic illness [introduction of unfamiliar environments]. Only 5% of those exposed to Legionella bacteria will develop Legionnaires' disease; 90% of those exposed will develop a less severe version of the infection called Pontiac fever (Heavey, 2016). Glover and Reed (2008) outlined the early development of prominent constitutional symptoms being malaise, lethargy, weakness, and anorexia. Infection of L. pneumophila initially involves a dry and nonproductive cough which progresses to one with produced mucus and sputum over several days (Glover and Reed, 2008). Along with non-productive coughing, the patient may commonly show signs of shortness of breath, and high fevers (which may exceed 40°C) with relative bradycardia as the illness progresses (Heavey, 2016) High body temperatures due to fever are a common onset with Legionnaires disease (Fields BS et.al, 2002). Chest pain and progressive dyspnea may be observed as well. Finally, extrapulmonary symptoms that would be evident during the course of the illness include diarrhea, nausea, and vomiting (Glover and Reed, 2008). Legionnaires disease can also be accompanied with myalgias (muscle pain) and arthralgias (joint pain), and possible changes in mental status (obtundation, hallucinations, seizures, and focal neurologic findings) (Glover and Reed, 2008). Gastrointestinal and neurologic manifestations of infection are shown particularly if the patient reports a history of hot tub use, travel, or recent hospitalization (Heavey, 2016). Interestingly, this aligns very well with Tom’s symptoms of vomiting and his use of hot tubs on the cruise.

Glover and Reed (2008) also stated that diagnostic tests are unavailable in many clinical laboratories, and therefore the diagnosis of Legionnaires’ disease is often presumptive, revolving around a suggestive clinical presentation. There is a general difficulty for diagnosis since the symptoms are very similar to other bacterial pneumonias (Heavey, 2016).

Legionnaires disease signs and symptoms including the timing of exposure and onset of illness.

The signs or objective characteristics noted by a healthcare professional for pneumonia include dyspnoea, cough, chills, chest pain, fever, decreased breath sounds, rales, rhonchi, and wheezes (Evertsen et al., 2010). These general signs of Legionnaires disease align with Tom’s signs that the healthcare professional noted on the cruise ship. The symptoms or subjective characteristics experienced by our patient, Tom, included sweating, coughing, headaches, muscles aches, and nausea. Coughing started roughly 5 days into the cruise trip. Clinical presentations for Legionnaires disease are typically observed from 2-10 days after the infection (Heavey, 2016). In accordance to the clinical symptoms outlined by Glover and Reed (2008), Tom could very well be suffering from Legionnaires disease- also presumed by Tom’s doctor in the case study presented for Week 3.


WORKS CITED:

1.Evertsen, J., Baumgardner, D. J., Regnery, A., & Banerjee, I. (2010). Diagnosis and management of pneumonia and bronchitis in outpatient primary care practices. Primary Care Respiratory Journal : Journal of the General Practice Airways Group, 19(3), 237–241. http://doi.org/10.4104/pcrj.2010.00024

2.Glover M.L., and Reed M.D., (2008). Chapter 111: Lower Respiratory Tract Infections. In: Dipiro JT, editor. Pharmacotherapy: Pathophysiological Approach. 7th edition. New York: McGraw Hill Medical. P1761-1790.

3.Fields BS, Benson RF, Besser RE. Legionella and Legionnaires’ Disease: 25 Years of Investigation. Clinical Microbiology Reviews. 2002;15(3):506-526. doi:10.1128/CMR.15.3.506-526.2002.

4.Heavey, E. (2016). Learning about Legionnairesʼ disease. Nursing, 46(8), 68-69. http://dx.doi.org/10.1097/01.nurse.0000484980.89786.7f Winn WC Jr. Legionella. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 40. Available from: https://www.ncbi.nlm.nih.gov/books/NBK7619/

Question 2

Which body system is effected. In what way has the normal physiological functioning of this area of the body been disturbed by the infection (without going into detail on the cause of this disturbance as this will be dealt with in questions 3 and/or 4). Representing this diagrammatically is always helpful. What made Tom susceptible to this infection?

Legionella pneumophila is a gram negative, opportunistic pathogen. It targets immunocompromised individuals and is a major nosocomial cause of infection. Infection occurs through breathing in aerosolized bacteria (Brusch, 2017). This results in the colonization and infection of the respiratory system. The normal physiological functions of the respiratory tract include respiration and bulk flow of oxygen and carbon dioxide and out of the body, respectively. Organs of the respiratory system include the nose, mouth, nasal cavity oral cavity, pharynx, larynx, trachea, bronchi, bronchioles, lungs and alveoli (American Lung Association). In particular, pneumonia targets the lungs. The lung parenchyma, which is the functional tissue of the lung, is made up of a large wall of alveoli, which provides a large surface area to support in effective gas exchange (Suki et. al, 2014). The alveoli are small sacs which are innervated by many capillaries and are the location for oxygen to enter the blood and carbon dioxide to leave the blood. The lungs also serve other essential functions such as monitoring and controlling the pH of the blood and maintaining blood pressure.

Figure 1: The components of the respiratory system

There are defence mechanisms such as mucus and nasal cilia throughout the respiratory system which work to keep bacteria and other pathogens out of your lungs, however in this scenario, these defense mechanisms did not work and led to the infection of the lungs by L. pneumophila. The alveoli of the lungs become inflamed as they are filled with fluids (American Lung Association). This results in difficulty of getting oxygen into the blood system for transport to cells and organs. A lack of oxygen can lead to decreased functioning of cells and organs, and if left untreated to death. Legionella pneumophila have displayed the ability to replicate within alveolar macrophages and alveolar epithelial cells (Newton, 1994). Additionally, there is evidence supporting that the macrophage monolayer within the respiratory system is destroyed at the peak of the L. pneumophila multiplication suggesting that L. pneumophila are involved in damage against alveolar macrophages (Nash et al., 1984).

Figure 2: The presence of fluid in the alveoli due to pneumonia

L. pneumophila multiplies in the alveolar macrophages and human monocytes often resulting in tissue damage (Rossier et. al, 2004 )(Jacobson et. al, 2008). Multiplying within macrophages allows it to avoid phagosome-lysosome fusion and the ability to transport around the body to deposit in different organs (Cunha et. al, 2016)(Brusch, 2017). It lives within a replicative phagosome inside macrophages to avoid vacuolar acidification and degradation from lysosomal enzymes (Brusch, 2017). It does this through inhibiting the phagocytic cells from forming a ribosome-lined replicative phagosome (Baltch et. al, 1998 ). Growth and replication of the bacteria can result in necrotic and hemorrhagic pneumonia within the lungs (Farver, 2018 ). Through cytopathogenicity, L. pneumophila can induce apoptosis in alveolar macrophages and epithelial cells (Gao and Kwaik, 1999).

Figure 3: L. pneumophila enters the macrophage and replicates. It then induces apoptosis in the cell to allow infection of neighbouring cells.

Invasion and constant replication in the lung parenchyma breaks down the immune defences and induces alveolar exudates to accumulate (Alcón et. al, 2005). Multiple lower lobes accumulate infiltrates and occasionally develop cavitation (Farver, 2018 ). Acute fibrinopurulent multinodular pneumonia usually occurs in the lungs (Skerrett et. al, 1989). The inflammatory alveolar exudate is filled with neutrophils, macrophages, monocytes, pus and fibrin (Farver, 2018 ). Edema also occurs at the site of infection. This inflammatory response is likely the cause of breathlessness in some individuals infected with L. pneumophila (Skerrett et. al, 1989). It may also decrease one’s ability to oxygenate blood and remove carbon dioxide. Reduced oxygen will cause the heart to work harder to increase blood flow and make up for the lack of oxygen thereby weakening the heart further. This will consequently decrease the flow of blood. Lack of blood flow to organs in the body, such as the kidney, can cause kidney failure. This results in the loss of the ability to filter waste out of the blood. Rhabdomyolysis, the break down of striated muscle cells causing increased myoglobin in the blood, is also occasionally caused by L. pneumophila and results in further kidney damage (Agu et. al, 2016).

The use of Tom’s corticosteroid nebulizer and the occurrence of asthma may have made him susceptible to this infection. Experiments were done on corticosteroid-treated rats to observe the effect on clearance of aerosolized Legionella pneumophila (Skerrett et. al, 1989). Corticosteroids were found to cause “a more diffuse pneumonitis, a persistent neutrophilic exudate, and a failure to recruit lymphocytes” (Skerrett et. al, 1989). Corticosteroids also inhibit the release of interleukin 1, which is required to activate lymphocytes (Skerrett et. al, 1989). It is speculated that the failure to recruit and activate lymphocytes, and the decreased number of neutrophils resulted in impaired expression of cell-mediated immunity. Middle aged (or older) men are at an increased risk of immune system suppression due to the use of corticosteroids (Glover et. Reed., 2008). With a suppressed immune system, Tom's body might not have been able to fight off an infection that it could have otherwise. Thus, increasing the susceptibility to L. pneumophila infection. Research also suggests that asthma can increase a person's susceptibility to infection by other respiratory pathogens since asthma also causes inflammation of the lungs (Young, 2014).

References:

1. Alcón, A., Fàbregas, N., & Torres, A. (2005). Pathophysiology of pneumonia. Clinics in Chest Medicine, 26(1), 39-46. 10.1016/j.ccm.2004.10.013.

2. Agu, C., Basunia, M., Salhan, D., Kandel, S., Schmidt, F., Quist, J., Enriquez, D. D49 LUNG INFECTION CASE REPORTS II: INFECTIONS OTHER THAN FUNGAL INFECTIONS: Legionella pneumonia associated with severe rhabdomyolysis and acute kidney injury. American Journal of Respiratory and Critical Care Medicine. 2016;193:1. https://search.proquest.com/docview/1863561416.

3. Brusch, J.L,. (2017). Legionnaire's disease: Cardiac manifestations. Infectious Disease Clinics of North America. 31(1):69-80.

4.Farver, C.F. (2018). Bacterial diseases - pulmonary pathology (second edition) - 10. Foundations in Diagnostic Pathology. 163–200. https://www-sciencedirect-com.ezproxy.library.ubc.ca/science/article/pii/B9780323393089000108.

5. Gao, L.Y., & Kwaik, Y.A. (1999). Apoptosis in Macrophages and Alveolar Epithelial Cells during Early Stages of Infection by Legionella pneumophila and Its Role in Cytopathogenicity. Infection and immunity, 67. Retrieved from: http://iai.asm.org/content/67/2/862.full (Links to an external site.)

6. Glover M.L., and Reed M.D., (2008). Chapter 111: Lower Respiratory Tract Infections. In: Dipiro JT, editor. Pharmacotherapy: Pathophysiological Approach. 7th edition. New York: McGraw Hill Medical. P1761-1790.

7. Learn About Pneumonia. American Lung Association. http://www.lung.org/lung-health-and-diseases/lung-disease-lookup/pneumonia/learn-about-pneumonia.html. Accessed 3 March 2018

8. Nash, T.W., Libby, D.M., & Horwitz, M.A. (1984). Interaction between the Legionnaires' Disease Bacterium (Legionella pneumophila) and Human Alveolar Macrophages: Influence of Antibody, Lymphokines, and Hydrocortisone. J. Clin. Invest., 47. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC425231/pdf/jcinvest00135-0111.pdf Accessed 3 March 201

9. Newton, H.J., Ang, D.K.Y., Driel, I.R., & Hartland, E.L. (2010). Molecular Pathogenesis of Infections Caused by Legionella pneumophila.N Clinical microbiology reviews, 23. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2863363/ Accessed 3 March 2018

10. Skerrett, S.J., Schmidt, R.A., Martin, T.R. (1989). Impaired clearance of aerosolized legionella pneumophila in corticosteroid-treated rats: A model of legionnaires disease in the compromised host. The Journal of Infectious Diseases. 160(2):261-273. http://www.jstor.org.ezproxy.library.ubc.ca/stable/30122890.

11. Suki, B., Stamenovic, D., & Hubmayr, R. (2014). Lung Parenchymal Mechanics. Compr Physiol., 1. Retrieved from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3929318/ (Links to an external site.)

12. Young, J. J. (2014). Risks for infection in patients with asthma (or other atopic conditions): Is asthma more than a chronic airway disease? Journal of Allergy and Clinical Immunology, 134(2), 258–259. doi:10.1016/j.jaci.2014.06.01 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4122981/ Accessed 3 March 2018

Question 3

Why did the doctor prescribe erythromycin and how does this antibiotic work to rid the body of the organism?

Erythromycin is a semisynthetic macrolide antibiotic used widely for many decades to treat mild-to-moderate bacterial infections caused by sensitive agents. Erythromycin is bacteriostatic against many gram positive bacteria including many strains of streptococci, staphylococci, clostridia, corynebacteria, listeria, haemophilus sp., moxicella, and Neisseria meningitidis (1). Since the doctor diagnosis Tom with Legionnaires , Erythromycin is a good therapeutic choice for Tom since he is experiencing a upper/lower respiratory infection. Also, this is a broad-spectrum antibiotic which is can be used to treat a variety of bacterial infections and it makes sense that the Cruise had the supply of it. Erythromycin, like all macrolide antibiotics, prevents bacterial cells from growing and multiplying by interfering with their ability to make proteins while not affecting human cells. In addition, the usual dosage for adults is 250 mg every 6 hours, 333 mg every 8 hours or 500 mg every 12 hours. Doses may be increased up to 4 g/day according to the severity of the infection. (2) Macrolides are a class of drug that has been in use as an antibiotic for more than 50 years. They typically function by binding to the bacterial ribosome. The 70S bacterial ribosome is made up of two subunits. The 30S ribosomal subunit binds messenger RNA, while the 50S ribosomal subunit binds tRNA. Inhibiting a necessary subunit of the ribosome prevents the bacteria from synthesizing protein. More specifically, macrolides bind at the peptidyl transferase center of the 50S subunit. This leads to the production of incomplete peptide chains, eventually causing cell death. (3) Two mechanisms of macrolide resistance, inducible expression of Erm methyltransferase and peptidemediated resistance, appear to depend on specific interactions between the ribosome-bound macrolide molecule and the nascent peptide. Macrolides accumulate within cells, suggesting that they may associate with receptors or carriers responsible for the regulation of cell cycle and immunity. (4)

Inhibition of Protein synthesis.gif

References

1, ERYTHROMYCIN (2018, January 18). In U.S National Library of Medicine. Retrieved March 10, 2018, from https://livertox.nih.gov/Erythromycin.htm

2. Kanoh, S., & Rubin, B. K. (2010, July). Mechanisms of Action and Clinical Application of Macrolides as Immunomodulatory Medications. NCBI, 23(3), 590-615.

3. Retsema, J., and Wenchi Fu. (2001) "Macrolides: Structures and Microbial Targets." International Journal of Antimicrobial Agents 18: 3-10. ]

4. Gaynor, M., & Mankin, A. (2003, May). Current Topics in Medicinal Chemistry. Bentham Science Publishers, 3(9), 949-960. 5. Image Reference Inhibition of protein synthesis by Antibiotics . http://chemistry.elmhurst.edu/vchembook/654antibiotic.html. Retreived March 9/2018

Question 4

Would this be considered an ‘outbreak’, is it reportable and to what official body. What role might the hot tub have played in this infectious scenario?

An ‘outbreak,’ according to the Centers for Disease Control and Prevention (CDC), is defined by the occurrence of more disease cases than would be expected in a give area considering a specific group of people over a particular period of time (Texier et al., 2016). Specifically, a Legionnaires’ disease outbreak can be labelled as an outbreak once there has been at least two people that have been exposed to Legionella (CDC: BC Centre for Disease Control, n.d.). The scenario here presented about Tom can be considered as a good example of a localized outbreak. A disease outbreak is reportable and should be given immediate attention.

However, one can only guess that this is an outbreak from the information that is given. Outbreaks of Legionnaires’ disease can sometimes be difficult to identify, especially if the location that one gets the disease from is not the hometown of all the individuals; they could be exposed during their travels and not develop any symptoms of the disease before arriving back home (CDC: BC Centre for Disease Control, n.d.). The best way to be sure it can be classified as an outbreak is if one refers to state and local health departments that will investigate the outbreaks and implement any control measures that are needed (CDC: BC Centre for Disease Control, n.d.).

Proper procedure should be followed to inform the Public Health Agency of Canada in order to execute the subsequent action against the disease. Created in 2004, the Agency the built to lead and act on public health matters that may arise (for example, national disease outbreaks and emergencies). In addition, the Agency has a lot of focus on health promotion, the prevention of chronic disease and injuries, and the response to public health emergencies. In hopes to improve electronic tools, the Agency was also involved in providing input in the designing of a pan-Canadian electronic tool called Panorama. Panorama allows health authorities to collect and manage health information to address public health issues like outbreaks (Public Health Agency of Canada, 2012).

If an outbreak were to occur in Canada, the front-line health care providers would probably be the first to know about the spread of disease. Which in this case presented, would be the hospital at the port. The doctor who initially examined Tom would only have known about Tom’s single diagnosis, but the hospital would be able to see the situation in greater magnitude, involving all the other passengers suffering from the disease. At the point in which the outbreak is first observed, it should be reported to the Public Health Agency of Canada immediately. In this instance, the reporting body would then design specific protocol to ensure the infection is contained and no further spread progresses. In addition, they should notify the specific area (or company in this case) in which the outbreak originated so proper sterilization and re-evaluation of hygienic measures are taken - which was done in this scenario through full scale sterilization regime of all hot tubs. Usually, outbreaks tend to happen when people encounter the infection in the same location at the same time, and it can easily be spread when a person breathes in small droplets of water in the air that is contaminated by Legionella (CDC: BC Centre for Disease Control, n.d.). Because of its relation to contaminated water, outbreaks of Legionnaires’ tend to be associated with structures that have complex water systems, just like the cruise ship that Tom developed the disease on (CDC: BC Centre for Disease Control, n.d.). It is most likely that Tom and other passengers on the ship got exposed to Legionella through water from showers, bathtubs, and other recreational water systems that are on the ship (CDC: BC Centre for Disease Control, n.d.).

The hot tub may have acted as a water reservoir for the infectious disease, pneumonia, when the first infected person used it (Kanamori, Weber, and Rutala, 2016). The bacteria may have resided in the hot tub environment until the next opportunity for infection came along. According to study done by Kanamori, Weber, and Rutala (2016), Legionella pneumophila can reside in bathing a tub immersion reservoirs and eventually cause pneumonia. This would then confirm the presumptive diagnosis of Tom’s doctor. Pneumonia can also be caused by Pseudomonas aeruginosa is also a bacteria known to cause pneumonia, and it can also use water environments as reservoirs (Kanamori, Weber, and Rutala, 2016).

Reference List:

[1] Kanamori, K., Weber, D., and Rutala, W.A., (2016). Healthcare Outbreaks Associated With a Water Reservoir and Infection Prevention Strategies, Clinical Infectious Diseases, Volume 62, Issue 11, 1. Pages 1423–1435, https://doi.org/10.1093/cid/ciw122

[2] Legionella - Outbreaks. (n.d.). In CDC: BC Centre for Disease Control. Retrieved from: https://www.cdc.gov/legionella/outbreaks.html

[3] Public Health Agency of Canada (2012). Website: http://www.phac-aspc.gc.ca/ep-mu/rido-iemi/index-eng. php#p

[4] Texier, G., Farouh, M., Pellegrin, L., Jackson, M. L., Meynard, J.-B., Deparis, X., & Chaudet, H. (2016). Outbreak definition by change point analysis: a tool for public health decision? BMC Medical Informatics and Decision Making, 16, 33. http://doi.org/10.1186/s12911-016-0271-x

Diagram of Legionnaires reservoir

The Microbiology Laboratory

Question 1

Other than the stated bacterial cause, what are the most common bacterial pathogens associated with this type of infectious scenario.

Background
Tom is experiencing a constellation of symptoms that point to a community-acquired pneumonia. Pneumonia is an inflammatory infection of the lungs, which primarily affects little air sac structures called ‘alveoli’. It can often lead to the accumulation of fluid in the alveoli, causing a wet cough. Other symptoms of pneumonia can range from nausea and fever, to chest pain and confusion. Given that Tom spent so much time in various hot tubs throughout the cruise ship and had recently experienced as asthma flare-up, his doctor diagnosed him with pneumonia, most likely caused by Legionella pneumophila. His symptom profile includes fever (indicated by sweating), a non-productive cough (indicating atypical pneumonia), as well as headaches, nausea, and myalgia. All of these symptoms are strikingly characteristic of pneumonia.
Hot tub exposure is an important clue in the diagnosis of this illness as Legionella pneumophila has a tolerance to heat and can exist in domestic or commercial water systems (1). Its risk to the public cannot be understated; by passing into water distribution networks from natural reservoirs (1), it contaminates water sources for homes, hotels, cruise ships, etc. Pneumonia from this bacterial species can arise when aerosolized particles of water are inhaled (1). Since hot tubs have a circulating and bubbling feature and are at a temperature such that some of the liquid water has vaporized, pulmonary exposure is highly likely.

Community-Acquired Pneumonia
Typical pneumonia presentation
Symptoms (2):
- rapid onset of fever and chills
- there is pleuritic chest pain
- cough (productive)
Signs (2):
- total leukocyte counts elevated
- C reactive protein (CRP) elevated
- Erythrocyte sedimentation rate (ESR) elevated
- Chest radiograph often showing lobar or segmental homogeneous opacity

Atypical pneumonia presentation
Symptoms (2):
- body aches
- fever (without chills)
- headache
- cough (unproductive)
Signs (2):
- total leukocyte counts normal to slightly elevated
- C reactive protein (CRP) normal to slightly elevated
- Erythrocyte sedimentation rate (ESR) normal to slightly elevated
- Chest radiograph often showing shadows (diffuse patchy or ground glass shadows)

Bacterial Species

Typical pneumonia
Species commonly associated with pneumonia include: Streptococcus pneumoniae (3) and Staphylococcus aureus which is a threat to those with chronic disease (4) like Tom's asthma coupled with inhaled corticosteroid therapy which has been found to be associated with an increased probability of pneumonia (5; 6; 7)
In one study on community-acquired pneumonia it was found that the most common bacterial cause of pneumonia was Streptococcus pneumoniae followed by Haemophilus influenzae, Chlamydia pneumoniae, Pseudomonas aeruginosa, and Mycoplasma pneumoniae (8).

Atypical pneumonia
The most common causative bacterial species for atypical community-acquired pneumonia are: Mycoplasma pneumoniae, Legionella pneumonophila (the presumptive diagnosis), and Chlamydia spp. (2).

Opportunistic bacteria
In those with obstructive pulmonary disorders, like asthma, it has been found that there is a disturbed microbiotic environment such that a higher frequency of pathogenic Haemophilus spp. are present (9), which could confer an increased pneumonia risk in the right immuno-environment. Because Tom's asthma had recently flared up and he had been taking inhaled corticosteroids, he was at a higher risk.
Chlamydia pneumoniae infection has been found to be associated with asthma, and is an opportunistic infection that is accountable for 10-15% of community-acquired pneumonia cases (10). Often, this bacteria is found in conjunction with others (coinfection) in community-acquired pneumonia – including M. pneumoniae and S. pnemoniae (10). M. Pneumoniae is likely to exacerbate asthma symptoms as well (11).
Finally, as there have been cases of pneumonia caused by P. aeurginosa as a result of exposure to contaminated hot tubs, this is also potentially a bacterial species of interest (12). Since P. aeruginosa is an opportunistic pathogen, and Tom was already chronically ill, had an asthma flareup and was concurrently on inhaled corticosteroids, he was more at risk to infection by an opportunistic bacteria.
Mycobacterium avium complex, or MAC, (comprised of M. avium and M. intracellulare or M. Fortuitum) can be transmitted via inhalation of aerosolized droplets containing the bacterial complex into the respiratory tract (13). This bacterial complex is associated with a hypersensitivity pneumonitis-like reaction (hot tub lung); this infection most often occurs in immunocompromised hosts (13). The symptoms of infection by MAC include cough, sputum production, weight loss, fever, lethargy, and night sweats (13). MAC and other non-tuberculous mycobacteria (NTM) can contaminate the hot tub water and patients infected by these pathogens express similar symptoms (14).



'Bacteria descriptions'


Streptococcus pneumoniae

Streptococcus pneumoniae

This bacteria is a gram-positive anaerobe, which can enter the lungs through either inhalation or the bloodstream. The bacteria is known to asymptomatically colonize the respiratory tracts and sinuses of healthy carrier(15). In immunocompromised individuals, however, the bacteria poses a greater risk of becoming pathogenic and establishing infection. S. pneumonia employs several virulent mechanisms in order to establish infection in the host, and produce symptoms characteristic of pneumonia. These include the bacteria's ability to firmly adhere to the alveolar epithelium, and secrete pore-forming toxic pneumolysin(15) which disrupts membrane integrity and leads to fluid accumulation in the lungs.

Staphylococcus aureus



Staphylococcus aureus
S. aureus is a gram-positive human pathogen that can cause a plethora of diseases, ranging in severity. Of the many infections mediated by this bacteria, pneumonia is one of the most common infections, and can potentially be life-threatening. S. aureus infections are extremely common, and often volatile, in a hospital environment. The bacteria are one of the leading etiologic agents of ventilator-associated pneumonia(16), and is being increasingly recognized as an important cause of community-acquired pneumonia- displaying the bacterias high capacity to infect a population of otherwise healthy individuals. One of S. aureus' key virulence factors, involved in establishing infection, is the production of the toxin alpha-hemolysin(16). This pore-forming cytotoxin is essential for pathogenesis, and strains lacking it are avirulent in many researched models. Although pneumonia caused by this bacteria can be extremely severe, the infection can be treated orally or intravenously with antibiotics. Despite this, there are an increasing number of resistant strains of S. aureus(16) which make treatment more challenging.

Haemophilus influenzae


Haemophilus influenza
Another common cause of bacterial pneumoniae is Haemophilus influenzae, a gram negative anaerobe(17) which was first described during an influenza pandemic. There are several different pathogenic strains of this bacteria, which can be either encapsulated or un-encapsulated. Amongst the encapsulated types, there exist 6 different serotypes of the bacteria with distinct capsular polysaccharide compositions. In regards to pneumonia, influenzae type b (Hib) is one of the most common infectious serotypes(18). The capsule can be considered one of the most important virulence factors of the bacteria, as it renders them increasingly resistant to phagocytosis(18) by leukocytes of the immune system. Since most strains of H. Influenzae are opportunistic, similar to S. pneumonia, the bacteria are usually only able to establish infections in individuals with weakened immune systems. This may include young (unimmunized) children, the elderly, or individuals with pre-existing medical conditions. In healthy individuals, however, the bacteria can be found colonizing the upper respiratory tract. Since the bacteria are commonly present in oral and nasal cavities(17), transmission usually occurs through direct contact with respiratory droplets released during sneezing and coughing.

Chlamydia pneumoniae


Chlamydia pneumoniae
Chlamydophila pneumoniae, a gram negative intracellular bacterium, is also spread from an infected individual to another person via coughing or sneezing. Unlike S. pneumoniae and H. influenzae, the incubation period of C. pneumoniae is much longer; it takes roughly 3 to 4 weeks following exposure for symptoms to present(19). This means that carriers of the bacteria may be contagious, and able to transmit the infection, for an extensive period of time. As a result, C. pneumoniae is another prominent infectious agent causing pneumonia. This extended incubation period, with a later onset of symptoms, may be viewed as a mechanism of virulence(19); the bacteria is able to multiply and spread without alarming the host, or others in contact with the host. Although most hosts are susceptible to infection, pathogenesis by C. pneumoniae is most commonly seen in school-aged children(19). Re-infections, however, are more common in older adults.


Pseudomonas aeruginosa




Pseudomonas aeruginosa
Pseudomonas aeruginosa is a gram negative bacteria which causes a range of diseases in plants and animals. Although less common, the bacteria is known to be an etiological agent of pneumonia and primarily infects immunocompromised hosts(20). Due to the increasing prevalence of hospital-acquired infections (such as ventilator pneumonia), the various types of infection which can result, and the emergence of multi-drug resistant strains(20), Pseudomonas is considered to be of extreme clinical relevance. The bacteria are able to thrive on warm, moist surfaces such as medical equipment (catheters, ventilators), hot tubs, and water pipe systems. It is this ability of the bacteria, to thrive in different environments, which readily enables transmission of the bacteria.

Mycoplasma pneumoniae




Mycoplasma pneumoniae
M. pneumonia is transmitted easily through contact with respiratory fluids, and is known to be extremely contagious. The bacteria is known to cause an atypical pneumonia, often referred to as 'walking pneumonia'(21), since it can spread quickly in crowded 'walking' areas such as schools, markets, and nursing homes. A dry cough is the most common sign of infection and, if left untreated, can progress to severe brain, heart, and kidney damage. The bacteria also establishes infection through the secretion of various cytotoxins, with Community Acquired Respiratory Distress Syndrome (CARDS) toxin(21) being of the the key virulence factors. Though diagnosis of M. pneumoniae is often difficult early on during the course of infection, the bacteria can be treated with antibiotics. Unfortunately the bacteria has been seen to persist even after treatment,which may be attributed to its ability to mimic host cell surface composition(21) and hide from/avoid killing by the hosts immune system.

Legionella pneumophila


Legionella pneumophila
In the case scenario, Tom's doctor likely suspects he has contracted Legionnaires disease (LD). LD is a form of atypical pneumonia caused by Legionella bacteria, with L. pneumophila being one of the most common etiological agents. L. pneumophila is an aerobic, gram-negative bacteria found naturally in fresh water(22). It can also, however, contaminate other moist environments such as hot tubs and cooling towers of air conditions. The prevalence, and severity, of infections caused by this bacteria may be attributed to its mechanism of pathogenesis.Upon transmission, the bacteria invade host macrophages and replicate within them(22). The prevalence of infection by L. pneumophila may be attributed to the bacterias ability to exploit host defence machinery during pathogenesis. Similar to other bacterial causes of pneumonia, the infection can be treated effectively with antibiotics such as tetracyclines, quinolines, and ketolides(22). The choice of antibiotic for treatment, however, requires careful consideration; since the bacteria multiple intracellularly, the antibiotic must be able to easily penetrate host cells in order for effective treatment.

Mycobacterium Avium Complex (MAC)



M. avium and M. intracellulare or M. Fortuitum
The mycobacterium avium complex (MAC) consists of a group of mycobacteria (mentioned above) which commonly establish infection in conjunction with one another. Since these bacteria are commonly found in food, water and soil(23), they are generally seen to establish infection in immunocompromised hosts. Individuals suffering from HIV, in particular, are extremely vulnerable to pathogenesis by the MAC. This group of bacteria are known to infect different parts of the body, including the lungs, bones or intestines(23). They can also cause systemic infections, which can be severe or even fatal. MAC has been identified as a contributing agent/ the causative agent of various respiratory diseases; this includes pneumonia, chronic obstructive pulmonary disease, chronic bronchitis and cancer. In general, MAC infection can be treated with 2 or 3 antimicrobial taken over the course of roughly 12 months(23). Commonly used drugs include macrolides, ethambutol and rifamycins. If left untreated, or in infectious scenarios with severely immunocompromised patients, can lead to death.




References 1. Leoni E, De Luca G, Legnani PP, Sacchetti R, Stampi S, Zanetti F. Legionella waterline colonization: detection of Legionella species in domestic, hotel and hospital hot water systems. Journal of Applied Microbiology. 2005 Feb 1;98(2):373.

2. Bedi RS. Community acquired pneumonia-Typical or atypical?. Lung India. 2006 Jul 1;23(3):130.

3. Murdoch DR, Laing RT, Mills GD, Karalus NC, Town GI, Mirrett S, Reller LB. Evaluation of a rapid immunochromatographic test for detection of Streptococcus pneumoniae antigen in urine samples from adults with community-acquired pneumonia. Journal of clinical microbiology. 2001 Oct 1;39(10):3495-8.

4. VanMeter KC, Hubert, RJ. GOULD'S Pathophysiology for the Health Professions. Canada: Elsevier Saunders; 2014.

5. Crim C, Calverley PM, Anderson JA, Celli B, Ferguson GT, Jenkins C, Jones PW, Willits LR, Yates JC, Vestbo J. Pneumonia risk in COPD patients receiving inhaled corticosteroids alone or in combination: TORCH study results. European Respiratory Journal. 2009 Sep 1;34(3):641-7. 

6. McKeever T, Harrison TW, Hubbard R, Shaw D. Inhaled corticosteroids and the risk of pneumonia in people with asthma: a case-control study. Chest. 2013 Dec 1;144(6):1788-94. 

7. Singh S, Amin AV, Loke YK. Long-term use of inhaled corticosteroids and the risk of pneumonia in chronic obstructive pulmonary disease: a meta-analysis. Archives of internal medicine. 2009 Feb 9;169(3):219-29. 

8. Ruiz M, Ewig S, Marcos MA, Martinez JA, Arancibia F, Mensa J, Torres A. Etiology of community-acquired pneumonia: impact of age, comorbidity, and severity. American journal of respiratory and critical care medicine. 1999 Aug 1;160(2):397-405.

9. Hilty M, Burke C, Pedro H, Cardenas P, Bush A, Bossley C, Davies J, Ervine A, Poulter L, Pachter L, Moffatt MF. Disordered microbial communities in asthmatic airways. PloS one. 2010 Jan 5;5(1):e8578. 

10. Hahn DL. Chlamydia pneumoniae, asthma, and COPD: what is the evidence?. Annals of Allergy, Asthma & Immunology. 1999 Oct 1;83(4):291-2. 

11. Nisar N, Guleria R, Kumar S, Chawla TC, Biswas NR. Mycoplasma pneumoniae and its role in asthma. Postgraduate medical journal. 2007 Feb 1;83(976):100-4.

12. Huhulescu S, Simon M, Lubnow M, Kaase M, Wewalka G, Pietzka AT, Stöger A, Ruppitsch W, Allerberger F. Fatal Pseudomonas aeruginosa pneumonia in a previously healthy woman was most likely associated with a contaminated hot tub. Infection. 2011 Jun 1;39(3):265-9. 

13. Koirala J. Mycobacterium Avium Complex (MAC) (Mycobacterium Avium-Intracellulare [MAI]). Medscape. [Online] November 14, 2017. https://emedicine.medscape.com/article/222664-overview.

14. Yasin H, Mangano WE, Malhotra P. Farooq A, Mohamed H. Hot Tub Lung: A Diagnostic Challenge. NCBI. [Online] Cureus, August 27, 2017. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5659329/.

15. Mitchell, A.m., and T.j. Mitchell. “Streptococcus pneumoniae: virulence factors and variation.” Clinical Microbiology and Infection, vol. 16, no. 5, 2010, pp. 411–418., doi:10.1111/j.1469-0691.2010.03183.x.

16. Ragle, B. E., et al. “Prevention and Treatment of Staphylococcus aureus Pneumonia with a -Cyclodextrin Derivative.” Antimicrobial Agents and Chemotherapy, vol. 54, no. 1, May 2009, pp. 298–304., doi:10.1128/aac.00973-09.

17.“Haemophilus influenzae.” Wikipedia, Wikimedia Foundation, 28 Feb. 2018, en.wikipedia.org/wiki/Haemophilus_influenzae.

18. “Haemophilus influenzae Disease (Including Hib).” Centers for Disease Control and Prevention, Centers for Disease Control and Prevention, 13 Feb. 2018, www.cdc.gov/hi-disease/clinicians.html.

19. Krüll, Matthias, and Norbert Suttorp. “Pathogenesis of Chlamydophila pneumoniae infections — epidemiology, immunity, cell biology, virulence factors.” Community-Acquired Pneumonia Birkhäuser Advances in Infectious Diseases, pp. 83–110., doi:10.1007/978-3-7643-7563-8_6.

20.“Pseudomonas aeruginosa.” Wikipedia, Wikimedia Foundation, 3 Mar. 2018, en.wikipedia.org/wiki/Pseudomonas_aeruginosa.

21. “Mycoplasma pneumoniae.” Wikipedia, Wikimedia Foundation, 6 Mar. 2018, en.wikipedia.org/wiki/Mycoplasma_pneumoniae.

22.Cunha, Burke A, et al. “Legionnaires disease.” The Lancet, vol. 387, no. 10016, 2016, pp. 376–385., doi:10.1016/s0140-6736(15)60078-2.

23. Ryu, Yon Ju, et al. “Diagnosis and Treatment of Nontuberculous Mycobacterial Lung Disease: Clinicians Perspectives.” Tuberculosis and Respiratory Diseases, vol. 79, no. 2, 2016, p. 74., doi:10.4046/trd.2016.79.2.74.

Question 2

What are all the samples that could be taken for laboratory testing (including the blood and ‘sputum’ in this case) and how important is the Microbiology Laboratory in the diagnosis of this particular infectious disease?

The clinical presentation, history, physical exam, and imaging results of legionnaire’s disease are non-specific and cannot be used to clinically distinguish legionella from other causes of pneumonia (1,2). The microbiology laboratory threrefore has an extremely important role in the diagnosis of legionnaire’s disease as there are a variety of bacterial, viral, and fungal pathogens that can all cause pneumonia. Each type of pathogen possesses different virulence factors, exhibit different structures, and are thus resistant to different types of treatment and therapy. The microbiology laboratory can determine the cause of the pneumonia, and subsequently, inform clinicians of effective treatment plans and recommendation of appropriate medication and antibiotics or anti-microbial agents. In addition, it is also important for distinguishing between pneumonia and other pulmonary conditions (Kubilay, Layon, Bae & Archibald, 2016). Furthermore, it is important that legionnaire’s disease be specifically tested for since the conventional laboratory work up for community acquired pneumonia would not identify legionella. Legionnaires’ disease cannot be definitively diagnosed without access to a lab (2,3). Determining the etiology of pneumonia is more important in developing countries, as the mortality and morbidity rates in these settings are quite high (Hammit et al., 2012). Conversely, in developed countries it can be argued that determining the etiology of pneumonia is not a cost-effective strategy, as some researchers have found that discovering the causative agent does not lead to changes in clinical management of the infection (Singh & Satinder, 2011).


Typical clinical specimens that may be taken for laboratory testing include: lower respiratory tract secretions, lung aspirates, pleural fluid, blood specimens, and urine specimens (Hammit et al., 2012; Murdoch et al., 2012). One possibly less common specimen that may be collected will also be discussed below.


1. LOWER RESPIRATORY TRACT (LRT) SECRETIONS: As pneumonia is an infection of the lower respiratory tract (LRT), it is imperative to collect and analyze secretions from the LRT. Sputum from the LRT is a very common specimen that is used in diagnosing pneumonia (Lahti et al., 2009). The risks associated with collecting sputum are low, and generally include coughing, wheezing, and vomiting (Hammit et al., 2012). Aside from sputum, bronchoalveolar lavage (BAL) may also be collected by instilling saline solution into the alveoli, followed by re-aspiration of the solution to collect the sample (Collins et al., 2014).


2. LUNG ASPIRATES: Collecting lung aspirates involves a multi-step procedure, wherein a needle is inserted percutaneously in the rib area, and aspirates are suctioned for about 2-3 seconds (Hammit et al., 2012). Although collecting these specimens is possible, it is usually not practical for the clinical laboratory.


3. PLEURAL FLUID: Pleural fluids are especially useful for cases of pneumonia that are complicated by pleural effusion (Hammit et al., 2012). Pleural effusion occurs when excessive fluid enters the pleural space, or the space outside of the lungs (Paramsivam & Bodenham, 2007).


4. BLOOD: Collection and testing of blood samples is a safe and routine method of diagnosing infectious disease (Hammit et al., 2012). Through serology, examining blood samples can inform clinicians of specific antibiotics that may be effective against the bacteria that is involved in the infection.


5. URINE: Urine samples may be used and analyzed through antigen detection tests in adults with suspected bacteria pneumonia (Hammit et al., 2012), and is especially useful for detecting Legionella antigen in adults (Andreo et al., 2006).


6. EXHALED BREATH CONDENSATE (EBC): EBC samples have previously been collected in a study comparing the protein spectrum of EBC in individuals with various lung diseases, including COPD, pneumonia, and non-small-cell lung carcinoma (Anaev et al., 2017). Presently, there is no standardized technique for collecting EBC’s, nor are there studies that have established biomarker values in EBC’s in patients with pneumonia (Hammit et al., 2012). However, investigators found that the EBC samples from different groups of donors exhibited characteristic protein spectrums, thus indicating the possible use of EBC in future diagnostic tests in the microbiology lab (Anaev et al., 2017).


SAMPLE COLLECTION, HANDLING AND TRANSPORT

Lower respiratory tract specimens and aspirates:

  • Timing: samples can be obtained at any time, but preferably before starting any antimicrobial treatment (CDC, Dec 2017).
  • Collection (BAL, pleural fluid, lung aspirates): specimens must be stored in sterile containers.
  • Collection (sputum): the patient will be asked to rinse their mouth. To collect the sample, a patient may be asked to cough and expectorate the sputum into a sterile container (Lagerstrom et al., 2004), or induced sample may be collected by inhaling saline vapours, which stimulates sputum production (Hammit et al., 2012).
  • Possibility of contamination: contamination of LRT specimens can commonly occur due to upper respiratory secretions (Murdoch, 2012). Even with thorough collection techniques, it is important to interpret laboratory results of these samples carefully.
  • Handling and transport: All vials and containers must be labeled with patient name, ID, type of specimen, and date collected. During transportation and storage, if the specimen can be plated within several hours, temperature should be set at 2 to 5 °C. Otherwise, long-term storage is optimal at - 70°C. It is important to note that freeze-thawing cycles should be avoided with respiratory tract specimen (Edelstein & Luck, 2015).


Blood:

  • Collection: small volume of blood samples following proper phlebotomy technique (Jeon et al., 2011).
  • Handling and transport: to reduce negative effects to protein stability and enzyme activity, long-term storage temperature should reach about −80 °C. This strategy may be used in Tom’s case, as there is a time delay between sample collection and reception at the hospital. To maintain this temperature, any frozen samples should be stored with dry ice. The total number of freeze-thaw cycles of specimens should be reduced for increased quality of the sample (Tuck et al., 2009).

If samples of whole blood plasma are required for PCR:

  • Timing: as soon as possible
  • Handling and transport: the sample should be kept in an EDTA tube. Specimens should be kept at 4 °C (transported on wet ice) (CDC, Dec 2017).


Urine:

  • Timing: urine samples should be collected within 7 days of onset of symptoms of pneumonia
  • Collection: the sample (10 – 20 mL) may be stored directly into sterile container
  • Handling and transport: All vials and containers must be labeled with patient name, ID, type of specimen, and date collected. Specimens should be kept at 4 °C (transported on wet ice) (CDC, Dec 2017). In addition, urine samples that have been frozen at -20 to -70 °C may still be used for other investigations, as they can remain stable for many months (Edelstein & Luck, 2015).


References:

UpToDate, "Community acquired pneumonia in adults" , 2017, Wolterz Kluver.

UpToDate, , 2017, and " Clinical manifestations and diagnosis of Legionella infection", 2017, Wolterz Kluver.

UpToDate, " Epidemiology and pathogenesis of Legionella infection", 2017, Wolterz Kluver.

Anaev, E., Kushaeva, M., Fedorchenko, K., Ryabokon, A., Kononikhin, A., Pikin, O., ... & Chuchalin, A. (2017). Diagnosis of lung diseases based on proteomic analysis of exhaled breath condensate.

Andreo, F., Domínguez, J., Ruiz, J., Blanco, S., Arellano, E., Prat, C., ... & Ausina, V. (2006). Impact of rapid urine antigen tests to determine the etiology of community-acquired pneumonia in adults. Respiratory medicine, 100(5), 884-891.

CDC. (December 2017). Unexplained Respiratory Disease Outbreaks (URDO): Specimen Collection and Handling. Centers for Disease Control and Prevention. Available from: https://www.cdc.gov/urdo/specimen.html and https://www.cdc.gov/urdo/downloads/SpecCollectionGuidelines.pdf

Collins, A. M., Rylance, J., Wootton, D. G., Wright, A. D., Wright, A. K. A., Fullerton, D. G., & Gordon, S. B. (2014). Bronchoalveolar Lavage (BAL) for Research; Obtaining Adequate Sample Yield. Journal of Visualized Experiments : JoVE, (85), 4345. Advance online publication. http://doi.org/10.3791/4345

Edelstein P, LÜCK C. 2015. Legionella, p 887-904. In Jorgensen J, Pfaller M, Carroll K, Funke G, Landry M, Richter S, Warnock D (ed), Manual of Clinical Microbiology, Eleventh Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817381.ch49

Hammitt, L. L., Murdoch, D. R., Scott, J. A. G., Driscoll, A., Karron, R. A., Levine, O. S., ... & Pneumonia Methods Working Group. (2012). Specimen collection for the diagnosis of pediatric pneumonia. Clinical infectious diseases, 54(suppl_2), S132-S139.

Kubilay, Z., Layon, A. J., Baer, H., & Archibald, L. K. (2016). When is pneumonia not pneumonia: a clinicopathologic study of the utility of lung tissue biopsies in determining the suitability of cadaveric tissue for donation. Cell and tissue banking, 17(2), 205-210.

Lagerström, F., Fredlund, H., & Holmberg, H. (2004). Sputum specimens can be obtained from patients with community-acquired pneumonia in primary care. Scandinavian journal of primary health care, 22(2), 83-86.

Lahti, E., Peltola, V., Waris, M., Virkki, R., Rantakokko-Jalava, K., Jalava, J., ... & Ruuskanen, O. (2009). Induced sputum in the diagnosis of childhood community-acquired pneumonia. Thorax, 64(3), 252-257.

Murdoch, D. R., O’Brien, K. L., Driscoll, A. J., Karron, R. A., Bhat, N., Pneumonia Methods Working Group, & PERCH Core Team. (2012). Laboratory methods for determining pneumonia etiology in children. Clinical infectious diseases, 54(suppl_2), S146-S152.

Paramasivam, E., & Bodenham, A. (2007). Pleural fluid collections in critically ill patients. Continuing Education in Anaesthesia, Critical Care & Pain, 7(1), 10-14.

Singh, V., & Aneja, S. (2011). Pneumonia–management in the developing world. Paediatric respiratory reviews, 12(1), 52-59.

Tuck, M. K., Chan, D. W., Chia, D., Godwin, A. K., Grizzle, W. E., Krueger, K. E., … Brenner, D. E. (2009). Standard Operating Procedures for Serum and Plasma Collection: Early Detection Research Network Consensus Statement Standard Operating Procedure Integration Working Group. Journal of Proteome Research, 8(1), 113–117. http://doi.org/10.1021/pr800545q

Question 3

Explain the tests that will be performed on the samples in order to detect any of the potential bacterial pathogens causing this disease.

Culture

Cultures are considered the gold standard for Legionella diagnosis and are commonly performed to confirm the presence of L. pneumophila. Aspirates, sputum, bronchoalveolar lavage fluid and pleural fluid all have the potential to contain Legionella bacteria and are therefore all appropriate samples to perform a culture on. As the species of bacteria causing infection, if any at all, is unknown, a Gram stain should first be done to confirm the presence of any Gram negative, small and filamentous rods, characteristic indicators of Legionella infection. (1). During this process, the chosen media should be liquefied, then cooled solified before subsequently adding the sample; notably, MacConkey agar is used commonly for Gram negative bacterial isolation. During incubation, the plate should be kept at room temperature away from direct light. (2).

Once a Gram stain has been completed and the species is confirmed to be Gram negative, samples should be incubated in a cell culture plate in buffered charcoal yeast extract and α-ketoglutarate. Importantly, this medium provides essential nutrients for Legionella growth, including iron and L-cysteine. The bacteria may also be grown on trypic soy blood agar, or buffered charcoal yeast extract lacking L-cysteine, as a control; lack of growth would support the hypothesis of Legionella presence as L-cysteine is essential for growth. (1). Optionally, the microbiologist may add antimicrobial agents to select against normal flora that or other bacteria that may be residing in the sample. The plate should be incubated at 35 °C for optimal growth conditions. Notably, longer incubation times are needed as Legionella is a slower-growing bacterium. (3) Results are often obtained within 3-5 days of plating, rendering this diagnostic test less convenient than quicker tests, such as DFA staining and the Urinary Antigen Test. Sensitivity ranges from 10-80%, and so may produce more false negatives and false positives than other more reliable testing methods. (4)

A legionella culture is performed here on black charcoal yeast extract with alpha-ketoglutarate.

In order to increase probability of Legionella recovery, selective pressures against normal flora that may be present in the sample can be introduced. Cycloheximide, polymyxin B and vancomycin are all toxic additives that do not inhibit Legionella growth but may hinder growth of other bacteria. Acid and heat exposure may also be used as Legionella is more tolerant of these environmental factors than some normal flora. (1).

Once a culture has been completed, a confirmation of L. pneumophila presence can be completed using matrix-assisted laser desorption ionization – time of flight (MALDI-TOF), a form of mass spectrometry. PCR may also be used to confirm the presence of genes specific to L. pneumophila. (4)  

PCR, Gel Electrophoresis and Sequencing

Polymerase Chain Reaction, or PCR, is used as a tool to amplify areas of the Legionella genome. (5) Many samples can be used for PCR analysis, including sputum, bronchioalveolar lavage fluid, serum and urine, as these samples may contain whole or lysed bacterial contents that can be used for diagnosis. Notably, this technique is favourable in that it does not necessarily require a sputum sample, which not all patients produce during infection. In this PCR reaction, primers to known Legionella genes of interest are added to a sample, along with a DNA polymerase to amplify the region of interest. The sample is heated to 94°C to denature the DNA. Then, temperature is reduced to 40-60 °C, an ideal temperature for primers to anneal to the single-stranded DNA. Temperature is once again raised to 70-74°C to facilitate extension of the new strand via the function of DNA polymerase. This process is repeated continuously until the regions of DNA have been amplified extensively and are at detectable levels. Positive and negative controls are also used for comparison of the level of PCR product to the experimental samples. (6) Primers prepared for Legionella PCR will often be prepared for Legionella ribosomal RNA genes, including 16S rRNA, 5S rRNA, and 23S rRNA, and the macrophage infectivity potentiator gene, or mip. (7) Advantageously, as mip is relatively conserved between serogroups, it can be used to detect Legionella serogroups 1-16. (8)However, because these genes have been reported in studies as having specificity of lower than 99% in PCR reactions, it is not often considered a reliable independent diagnostic test, and may be coupled with other tests for confirmation. (6)

The results can be visualized using gel electrophoresis. PCR product is run through an agarose gel for 2-3 hours, and are then visualized under UV light in order to detect the amount of PCR product. Agarose gel is advantageous in that it is more porous than polyacrymide gel. As large amounts of DNA fragments are produced during PCR, this large pore size allows molecules to run quickly through the gel so that a size estimate of the genes can be reached.(9) Bands are produced on the gel based on size, with the smallest sized products moving further down the gel due to decreased resistance. Thus, the base pair length of the genes of interest must be known in order to confirm that the gene of interest has been amplified. (10)

Nucleotides are added to the primers after denaturing of DNA to allow an amplification of the strand of interest. This process is repeated until many copies of Legionella genes have been created.

To confirm the presence of Legionella DNA, the DNA is sequenced. Sanger sequencing methods are more likely to be used in this case than next generation sequencing as only one fragment is being amplified. It is then compared to Legionella genes located in databases such as NCBI BLAST. BLAST contains a database of known genes in strains of organisms, and allows one to infer homology based on the alignment and overlap of sequenced genes to genes in the database.

Direct Fluorescent Antibody (DFA)

Fluorecein-labeled antibody attached to Legionella bacilli

     This rapid microscopic procedure is used after microbial cultures and is not used as a single method to identify legionella due to its possibility of false positive and false negative results (11). Specimens used for this method are sputum, respiratory secretions, tissue or blood. Samples are placed on a plate on circles of 10mm in length, placed under a microscope, and incubated with antibodies for various serogroups. In this method monoclonal antibodies specific for Legionella species and serogroups are tagged with a fluorescent dye to illuminate a target antigen. The advantages for this method are that it is inexpensive and the commercially available reagents for identification and typing. The disadvantages for this method are that cross reactivity may complicate interpretations and it is technically demanding (11).

Urinary Antigen Test (UAT): EIA/ELISA

This testing laboratory method is highly used in Europe and the United States (11). The advantages for this method are the availability of the sample, quick speed and it gives positive results for long periods even after antibiotic treatment. Shortly after clinical symptoms appear (2-3 days) Legionella specific urinary antigens can be detected and excreted for many days to about 10 months, even during antibiotic treatment (11). Legionella UAT is available from many vendors in two formats: a 96-well plate-based enzyme immunoassay (EIA) or an ELISA, and a immunochromatographic test (ICT), in card or strip based formula (11). ELISAs for detecting urinary antigen against multiple Legionella have been tested with some success but are not yet commercially available (12). For detection of Legionella pneumophila serogroup 1, urinary antigen tests have sensitivities in the range of 70%-100% - so are used in areas with this serogroup is the main cause of the disease (13). The test cards include a membrane coated with rabbit antibody specific for Legionella pneumophilla serogroup 1 antigen and with controlled antibody is combined with rabbit anti-Legionella pneuphila serogroup 1 antigen + antispecies conjugates in a hinged test card (14).

Serological Testing and Antibody-Based Assays

Indirect fluorescent antibody test is the most common which detects immunoglobulin IgG, IgM and IgA antibodies (12). Micro-ELISA , counterimmunoelectrophoresis and microagglutination techniques have similar efficacy (12). Due to the high amount of antibodies in many individuals this limits the usefulness of getting a single acute titer (12).  ELISA kits which are specific for L. pneumophila IgM and IgG are used to identify serum samples with amounts of anti- L. pneumophila immunoglobulins. Lipopolysaccharide from L. pneumophila are plated, serum is isolated from centrifuged blood, diluted and then incubated for 90 minutes in the plate then it is washed. Then immunofluorescent tags specific to the antibodies are added. DFA assays, slide agglutination tests (SAT’s) and Mab screens are antibody based and are useful for qualitative Legionella identification, typing at the species and serogroup levels. Slide agglutination is used with a culture isolate, its advantages are that it is commercially available and inexpensive. Slide agglutination disadvantages are that cross – reactivity may complicate interpretation and it requires a culture isolate. MAb blotting (monoclonal antibody) also requires a culture isolate and it is a simple procedure for L. pneumophila serotype 1. MAb blotting’s disadvantages are that it is limited in availability and that it requires a culture isolate.

References

(1) Jorgensen, J. H., & Pfaller, M. A. (Eds.). (2015). Manual of clinical microbiology. Retrieved from http://ebookcentral.proquest.com

(2) Garcia, L. S. & Isenberg, H. D. (2010). Clinical Microbiology Procedures Handbook, Volumes 1-3 (3rd Edition). American Society for Microbiology (ASM). Available from: https://app.knovel.com/hotlink/toc/id:kpCMPHVE03/clinical-microbiology/clinical-microbiology

(3) Bram M. W. Diederen, Jan A. J. W. Kluytmans, Christina M. Vandenbroucke-Grauls, Marcel F. Peeters. Utility of real-time PCR for diagnosis of legionnaires’ disease in routine clinical practice. Journal of Clinical Microbiology. 2008;46(2):671-677. http://jcm.asm.org/content/46/2/671.abstract. doi: 10.1128/JCM.01196-0

(4) McGrath B, Drake J. Bronchoscopy in the ICU: An overview of broncho-alveolar lavage and bronchial washing  Airway eLearning.2014. http://www.airwayelearning.com/awel/articles/articles-1.aspx?Action=1&NewsId=2115&M=NewsV2&PID=71655.

(5) Chen DJ, Procop GW, Vogel S, Yen-Lieberman B, Richter SS. Utility of PCR, culture, and antigen detection methods for diagnosis of legionellosis. Journal of clinical microbiology. 2015;53(11):3474-3477. http://www.ncbi.nlm.nih.gov/pubmed/26292304. doi: 10.1128/JCM.01808-15.

(6) Den Boer J, Yzerman E. Diagnosis of legionella infection in legionnaires’ disease. Eur J Clin Microbiol Infect Dis. 2004;23(12):871-878. http://www.ncbi.nlm.nih.gov/pubmed/15599647. doi: 10.1007/s10096-004-1248-8.

(7) PCR amplification. Promega Web site. https://www.promega.ca/resources/product-guides-and-selectors/protocols-and-applications-guide/pcr-amplification/. Updated 2018.

(8) Nazarian, E. J., Bopp, D. J., Saylors, A., Limberger, R. J., & Musser, K. A. (2008). Design and implementation of a protocol for the detection of Legionella in clinical and environmental samples. Diagnostic microbiology and infectious disease, 62(2), 125-132

(9) Stellwagen NC. Electrophoresis of DNA in agarose gels, polyacrylamide gels and in free solution. Electrophoresis. 2009;30(Suppl 1):S188-S195. doi:10.1002/elps.200900052.

(10) D S Lindsay, W H Abraham, R J Fallon. Detection of mip gene by PCR for diagnosis of legionnaires' disease. Journal of Clinical Microbiology. 1994;32(12):3068-3069. http://jcm.asm.org/content/32/12/3068.abstract.

(11) Mercante, J. W., & Winchell, J. M. (2015). Current and Emerging Legionella Diagnostics for Laboratory and Outbreak Investigations. Clinical Microbiology Reviews, 28(1), 95–133. http://doi.org/10.1128/CMR.00029-14

(12) Kim, M. J., Sohn, J. W., Park, D. W., Park, S. C., & Chun, B. C. (2003). Characterization of a Lipoprotein Common to Legionella Species as a Urinary Broad-Spectrum Antigen for Diagnosis of Legionnaires’ Disease. Journal of Clinical Microbiology, 41(7), 2974–2979. http://doi.org/10.1128/JCM.41.7.2974-2979.2003

(13) David R. Murdoch, L. Barth Reller, Melvin P. Weinstein,; Diagnosis of LegionellaInfection, Clinical Infectious Diseases, Volume 36, Issue 1, 1 January 2003, Pages 64–69, https://doi.org/10.1086/345529 (Links to an external site.)

(14) Alere (2018). Alere BinaxNOW Legionella Urinary Antigen Card. Retrieved from https://www.alere.com/en/home/product-details/binaxnow-legionella.html (Links to an external site.)

Question 4

What are the results expected from these tests allowing for the identification of the bacteria named in this case.

Culture

en:Legionella sp. colonies growing on an en:agar plate and illuminated using ultraviolet light to increase contrast

Black Charcoal Yeast extract with alpha-ketoglutarate formulations are routinely used in clinical microbiology with the presumptive diagnosis of Legionnaires’ disease. The bacteria is observed over a 3-5 day period, where colonies are 0.5 – 1 mm in diameter and most colonies are smooth, glistening, and convex. The colonies are mostly white but can have blue, green, or red coloration (1). The longer the incubation, the morphology will change into a more filamentous and whiter coloration, but under ultraviolet light they will fluoresce with yellow/green coloration (2). L. micdadei and L. maceachernii will appear blue since it takes up the bromothymol blue dye and L. pneumophila will appear green (3). L. bozemanii is white-gray to blue-gray and will fluoresce blue-white under long wave UV light (4). In the late stage colonies, it is hard to distinguish between Legionella and non-Legionella species so daily observation is crucial (5).

PCR, Gel Electrophoresis and Sequencing

Pulsed-field gel electrophoresis patterns for Legionella pneumophila

DNA from the samples that Tom provides can be amplified through PCR to rapidly detect the presence of Legionella. By comparing DNA fragment sizes of a known size against the DNA ladder, it can confirm the Legionnaires’ disease diagnosis (12). Results can also be measured in threshold values, so when primers, specific to the genes of interest, are added, the threshold value is reached earlier if the genes are in abundance. If the threshold is reached before 50 cycles, it means L. pneumophila is detected. Depending on the primers added for specific genes, the microbiologist would expect to see an early threshold cycle for Legionella 16S, 5S, and 23S rRNA, as well as for the mip gene (13). Gene sequencing can confirm the PCR products correspond to the gene of interest if it is similar to that of the database sequence which can be provided by NCBI BLAST. NCBI BLAST indicates a score for likelihood of alignment, with a higher score indicating higher alignment, it also provides an E-value which signifies the likelihood that another sequence could get the same score by random chance. The lower the E-value with a high alignment score, the higher the homology likelihood, which can help confirm pathogen diagnoses (14).

Direct Fluorescence Antibody Staining (DFA Staining)
Legionella DFA

Direct fluorescent antibody tests use monoclonal antibodies to illuminate a target antigen from sputum samples (6). A microscope assesses the results of the direct immunofluorescence examination after unbound antibodies are rinsed off, usually in 2-4 hours (7) . If fluorescing rods are seen they are indicative of bacterial presence (≥ 25 rods is considered a positive result for Legionella infection) (8).

Urinary Antigen Test (UAT) - EIA/ELISA

Alere BinaxNOW® Legionella Urinary Antigen Card

L. pneumophila serotype 1 is detected with the urinary antigen tests as their sensitivities are 70-100% (7). The UAT uses EIA and ICT, which are based on the lateral flow assay, or LFA test strip or card, which contain:

• Sample pad: the sample, urine, will be placed here with a swab

• Conjugate release pad: the sample will flow towards this pad

• Membrane: the section of the card that has a “test line” and “control line”; this is the area that is inspected to determine the presence or absence of antigen from the sample. When no sample is present, no lines are visible on the membrane.

• Test line: includes antibodies against specific antigen

• Control line: lacks any antibodies against specific antigen

Commercial kits use enzyme immunoassay as well as immunochromatographic assay to detect with similar sensitivity and specificity. A positive specimen will give two pink-purple coloured lines, indicating that antigen was detected, any visible line is positive. A negative specimen will give a single coloured pink-purple control line in the top half of the window, meaning that no L. pneumophila serogroup 1 antigen was detected. (9). This occurs because the antibody on the test strip forms an antibody-antigen complex that forms the sample line which can be compared to a control line. Results are provided in 15 minutes and can prove L. pneumophila serotype 1 is the cause of the symptoms when it detects soluble antigens pertaining to serotype 1 (7). The colloidal gold turns pink if it is positive (10).

Serological Tests
Schema for identification ofLegionella organisms. BCYE, Bufferedcharcoal yeast extract; LRT, lower respiratory tract; DFA, direct fluorescent antibody.

Similar to the urinary antigen test, plate wells are read on a plate reader to determine the fluorescent concentration. A higher fluorescence level is expected for patients infected with L. pneumophila since antibodies will be present. It is important to note that cross-reactivity of other antibodies with L. pneumophila is possible (7). Antibody levels tend to be significantly lower in older adults due to a general weakening of the immune system with age, suggesting that other methods may produce more reliable diagnostics for Tom (11). If there is a reaction when the serum is mixed with the known antigen of L. pneumophila, then there is a presence of antibodies to L. pneumophila, which means the patient has the disease. This can also tell us the amount of titer to see how treatment is working (16).

References 1. LÜCK, C and Edelstein, P H. Manual of Clinical Microbiology, Eleventh Edition . 2015.

2. Isolation of Legionella spp. from environmental water samples by low-pH treatment and use of a selective medium. Bopp, C A, et al. 4, 1981, Vol. 13.

3. Nosocomial Legionella micdadei infection in transplant patients: fortune favors the prepared mind. Muder, R R, Stout, J E and Yu, V L. 4, 2000, Vol. 108.

4. Hardy Diagnostics. BUFFERED CHARCOAL YEAST EXTRACT (BCYE) AGAR. IFU Instructions for Use. [Online] Hardy Diagnostics .

5. Manual of clinical microbiology. Jorgensen, J. H., & Pfaller, M. A. 2015.

6. Parker, N, et al. Microbiology. Houston : OpenStax, 2016.

7. Diagnosis of Legionella Infection. Reller, L B, Weinstein, M P and Murdoch, D R. 1, s.l. : Clinical Infectious Disease, 2003, Vol. 36.

8. Test Evaluation. Joselson , R A. s.l. : Lab Med UCSF.

9. Alere. Alere BinaxNOW® Legionella Urinary Antigen Card. Alere. [Online] 2018. https://www.alere.com/en/home/product-details/binaxnow-legionella.html.

10. Rapid Diagnosis of Legionnaires' Disease Using an Immunochromatographic Assay for Legionella pneumophila Serogroup 1 Antigen in Urine during an Outbreak in The Netherlands. Weaver, P C, et al. 7, s.l. : Journal of Clinical Microbiology, 2000, Vol. 38.

11. Value of Serological Testing for Diagnosis of Legionellosis in Outbreak Patients. Rojas, A, et al. 8, s.l. : Journal of Clinical Microbiology, 2005, Vol. 43.

12. The simple and rapid detection of specific PCR products from bacterial genomes using Zn finger proteins. Osawa, Y, et al. 11, s.l. : Nucleic Acids Research, 2008, Vol. 36.

13. Evaluation of real-time PCR for the early detection. Diederen, B M, et al. s.l. : Journal of Medical Microbiology, 2007, Vol. 56.

14. Murphy, Michael. Lecture 9 Sequence Similarity Searching. MICB 325 Analysis of Microbial Genes and Genomes. Vancouver : s.n., 2018.

15. Direct Fluorescent Antibody Test. Case Studies in Microbiology. [Online] https://courses.cit.cornell.edu/biomi290/microscopycases/methods/fabs.htm.

16. Cunha BA. Legionnaires Disease. Medscape. [Online] June 9, 2017. https://emedicine.medscape.com/article/220163-overview.

17. Kaiser, G. Lab 17: Serology, Direct and Indirect Serologic Testing. LibreTexts. [Online] March 24, 2016. https://bio.libretexts.org/Labs/Microbiology_Labs_II/Lab_17%3A_Serology%2C_Direct_and_Indirect_Serologic_Testing.

Bacterial Pathogenesis Questions

Using the following pathogenic steps outline the pathogenesis of the bacteria named as being responsible for this infection.

Question 1

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. How would our patient have come in contact with this bacteria

Naturally occurring aquatic Legionella pneumophila can be found in freshwater environments such as rivers and lakes (1). They grow in amoebae and ciliated protozoa, including Hartmanella, Acanthamoeba and Naeglaria species (2), which provide protection against the environment and are similar to alveolar macrophages in the human host, which allows the bacteria to grow and multiply in during human infection (3). L. pneumophila can travel great distances in freshwater environments and have been detected over 3km away from their upstream source. This bacteria can be found globally, surveillance shows that incidence of Legionella disease occurs in the USA, Canada, New Zealand, Australia, Japan, Singapore, and in Europe. (13) It’s difficult to accurately state the epidemiology of L. pneumophila in other countries, due to lack of proper surveillance systems, diagnostics, and reporting. A 2014 study states high numbers of cases in France, Italy, Spain, and increasing incidence in the USA. (13) It has also been found responsible for nosocomial infections. L. pneumophila is more common in developed countries which contain greater numbers of complex water systems which provide opportunities for proliferation (1).

An outbreak of L. pneumophila in 2003 was linked to potting soil. A study by the School of Public Health at La Trobe University in Australia found a plant nursery that was using untreated river water to water plants (6). The river water was contaminated, and this resulted in the nursery selling plants and contaminated products. This demonstrates that L. pneumophila can also survive for several days on warm moist soil.

The optimal growing temperature for L. pneumophila is 35C, but they can grow at temperatures between 20C and 50C (1). Therefore, hot tubs and pools can provide optimal conditions for this bacteria to proliferate. Hot tubs are often linked to outbreaks in L. pneumophila, which appears to be the pathogen source in this case. High temperatures in hot tubs cause evaporation, and when this is combined with large numbers of people bathing, disinfectant levels become depleted. Bacteria that grow under these conditions are known as Recreational Water Illnesses (RWIs) (11). The primary causes of bacterial outbreaks in hot tubs are uncontrolled water temperatures, insufficient levels of disinfectants, and lack of cleaning leading to the formation of biofilms. Ideally, cold water should be kept below the temperature allowing for L. pneumophila growth, and hot water should be kept above the growth temperature. Furthermore, hot tubs are a significant source of aerosols which facilitates entry into the host. These outbreaks can often be linked back to a larger source of water, such as the cooling tower on the cruise ship. Aerosols and droplets with L. pneumophila are spread from some common artificial water systems, including faucets, building air-conditioning units, hot tubs not drained after use, water fountains, water tanks and heaters, and more.The key to prevention in many of these cases is avoiding stagnation which permits the formation of biofilms (7). Less commonly, people can be infected by having contaminated drinking water enter their respiratory tract, either through accidental inhalation or due to swallowing difficulties. (5)

Another outbreak in 2015 was linked to street cleaning trucks in Spain. The primary case was a man who worked on a street cleaner using high pressure water. The water used in the truck was drawn from untreated groundwater. These high pressure sprays created aerosols which were the most likely candidate for L. pneumophila transmission (12).

In the various aquatic environments L. pneumophila can encounter with differing temperatures, pH, nutrient availability, etc. the bacteria copes with stressful conditions by entering a temporary noncultivable state, where cell division is decreased, but metabolic activity is maintained, until favorable conditions return. (7) L. pneumophila can also form biofilms to provide protection when outside a protozoan host, also using them to create nutrient gradients. Additional strategies include a type II secretion system that produces effectors that allow L. pneumophila to obtain nutrients and survive stressful conditions such as oxidative stress or the presence of bactericidal/bacteriostatic molecules from other microbes (7). L. pneumophila has genes that are necessary for survival and replication in both amoeba and host macrophage cells, such as lpg0730 and lpg0122 that encode parts of an ATP binding cassette (ABC) transport complex, with unknown function as of yet (10).

  1. Legionellosis. (2017, November). Retrieved from http://www.who.int/mediacentre/factsheets/fs285/en/
  2. Hoffman, P. S. (1997). Invasion of Eukaryotic Cells byLegionella Pneumophila: A Common Strategy for all Hosts? Canadian Journal of Infectious Diseases, 8(3), 139-146. doi:10.1155/1997/571250
  3. Legionella | Clinical Disease Specifics | Legionnaires | CDC. (2017, December 7). Retrieved from https://www.cdc.gov/legionella/clinicians/disease-specifics.html
  4. Phin, N., Parry-Ford, F., Harrison, T., Stagg, H. R., Zhang, N., Kumar, K., … Abubakar, I. (2014). Epidemiology and clinical management of Legionnaires' disease. The Lancet Infectious Diseases, 14(10), 1011-1021. doi:10.1016/s1473-3099(14)70713-3
  5. Legionella | Causes and Transmission | Legionnaires | CDC. (2017, September 14). Retrieved from https://www.cdc.gov/legionella/about/causes-transmission.html
  6. Winn WC Jr. Legionella. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 40. Available from: https://www.ncbi.nlm.nih.gov/books/NBK7619/
  7. Ohno, A., Kato, N., Yamada, K., & Yamaguchi, K. (2003). Factors Influencing Survival of Legionella pneumophila Serotype 1 in Hot Spring Water and Tap Water. Applied and Environmental Microbiology, 69(5), 2540-2547. doi:10.1128/aem.69.5.2540-2547.2003
  8. Murga, R., Pruckler, J. M., Forster, T. S., Donlan, R. M., Brown, E., & Fields, B. S. (2001). Role of biofilms in the survival of Legionella pneumophila in a model potable-water system. Microbiology, 147(11), 3121-3126. doi:10.1099/00221287-147-11-3121
  9. Soderberg, M. A., Dao, J., Starkenburg, S. R., & Cianciotto, N. P. (2008). Importance of Type II Secretion for Survival of Legionella pneumophila in Tap Water and in Amoebae at Low Temperatures. Applied and Environmental Microbiology, 74(17), 5583-5588. doi:10.1128/aem.00067-08
  10. Lama, A., Drennan, S. L., Johnson, R. C., Rubenstein, G. L., & Cambronne, E. D. (2017). Identification of Conserved ABC Importers Necessary for Intracellular Survival of Legionella pneumophila in Multiple Hosts. Frontiers in Cellular and Infection Microbiology, 7. doi:10.3389/fcimb.2017.00485
  11. United States Department of Health and Human Services. “What Owners and Managers of Buildings and Healthcare Facilities Need to Know about the Growth and Spread of Legionella.” Https://Www.cdc.gov/Legionella/Water-System-Maintenance/Growth-and-Spread.html, 14 Sept. 2017, www.cdc.gov/legionella/water-system-maintenance/growth-and-spread.html.
  12. Valero, Natalie. “Street Cleaning Trucks as Potential Sources of Legionella Pneumophila.” Centers for Disease Control and Prevention, Nov. 2017, wwwnc.cdc.gov/eid/article/23/11/16-1390_article.
  13. Phin, N., Parry-Ford, F., Harrison, T., Stagg, H. R., Zhang, N., Kumar, K., … Abubakar, I. (2014). Epidemiology and clinical management of Legionnaires' disease. The Lancet Infectious Diseases, 14(10), 1011-1021. doi:10.1016/s1473-3099(14)70713-3

Question 2

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.

The pathogenesis of Legionella infections begins with the inhalation of contaminated aerosols. The only other mechanism of entry is through superficial wounds but that is very rare. Usually, Legionella organisms are cleared out of the upper respiratory tract through mucociliary action. Individuals with chronic diseases like lung disease or diabetes or have a weakened immune system have compromised mucociliary clearance and therefore, have a higher risk of Legionella infection. In immunocompromised individuals, L. pneumophilia may not all be cleared by mucociliary clearance and will be able to travel down to the lower respiratory tract and induce infection. In the case study, Tom’s asthma condition compromises his body’s ability to clear the bacteria and ultimately facilitates the infection of Legionella(1).

The ability of Legionella pneumophila to multiply intracellularly is its primary feature however, there is a lack of knowledge regarding exactly how the bacteria are internalized by eukaryotic cells as well as released to infect other cells(2). Nevertheless, there are a number of bacterial factors that have been identified to play a role in the attachment and entry of the bacteria into host cells, including, EnhC, Lcl, Hsp60, MOMP, type IV pili, LpnE, RtxA, and LadC(2).

EnhC is a periplasmic protein which plays an important role for efficient replication of the bacteria in macrophages(3). It also carries the responsibility to maintain cell wall integrity(2). EnhC does not play a direct role to bacterial invasion however, it does reduce Nod1 of the host cell which decreases innate immune recognition of the bacteria(2).

Hsp60 is one of the most abundant proteins synthesized by the bacteria, especially during growth that takes place in a variety of eukaryotic host cells(2). This protein is able to mediate phagocytosis of by modulating the function of macrophages, playing an important role in bacterial entry(2).

MOMP plays a large part in the attachment of Legionella pneumophila to host cells(2). It has been found that complement receptors, CR1 and CR3, of human monocytes mediates phagocytosis of the bacteria however, the fixing of C3 to the bacterial surface by the alternative pathway of the complement system is mediated by MOMP(2). This ultimately leads to a more convenient entry for the pathogen into the host cells(2).

Type IV pili is involved in both attachment and entry of the bacteria into host cells(2). Type IV pili’s most common function involves adherence of the bacteria to host tissues, facilitating bacterial invasion(3). It also plays a role in the formation and development of biofilms, which promotes the adherence of the pathogen and facilitates the survival of bacterial cells in environmental conditions that may be variable(2).

LpnE plays a contributing role in the mediation of Legionella pneumophila attachment to host cells(2). Its encoding gene, the IpnE gene, was found to be required for full entry of the bacteria into macrophage and further studies demonstrated that LpnE was required for efficient infection(2). In addition, LpnE had an influencing role in the trafficking of the Legionella pneumophila vacuole, which suggests that it may have interactions with eukaryotic cell proteins(2).

RtxA seems to play a role in bacterial attachment and entry however its mechanisms are still yet to be known. However, mutants without RtxA resulted in diminished attachment and adherence of the bacteria to human cells(2). Lcl and LadC are also proteins that have been identified to contribute to adherence, and invasion of host cells(2).

Ultimately, through complement receptors, bacteria bind to alveolar macrophages of the host, and are then engulfed into a phagosomal vacuole(3).

REFERENCES 1. Rathore, M.H. (November 02, 2017). Legionella infection. Retrieved February 28, 2018, from https://emedicine.medscape.com/article/965492-overview#a5

2. Zhan, X., Hu, C., & Zhu, Q. (2015, July 27). Legionella Pathogenesis and Virulence Factors. Retrieved

   March 02, 2018, from http://www.aclr.com.es/clinical-research/legionella-pathogenesis-and-virulence-
   factors.php?aid=6682

3. Winn, W. C., & J. (1996, January 01). Legionella. Retrieved February 27, 2018, from https://

   www.ncbi.nlm.nih.gov/books/NBK7619/#A2222

Question 3

Multiplication and Spread: does the organism remain extracellular or do they enter into cells and what are the molecular and cellular determinants of these events. Do the bacteria remain at the entry site or do they spread beyond the initial site i.e. are there secondary sites of infection and why do the bacteria hone in on these particular secondary sites.

Engulfment:

The organism is an intracellular pathogen for its replication phase. L. pneumophila can multiply intracellularly in human macrophages, including lung alveolar macrophages. After being phagocytosed, the organism inhibits the oxidative burst, and reduces phagosome acidification. (Horwitz 1983) The phagosome is used by the bacteria for the site of replication.

Legionella’s life cycle within the macrophages.

Macrophage provides a permissive environment for the bacteria to grow and replicate. When the bacteria entered the vacuole, it altered its composition as explained above, for instance the usual organelle trafficking is modified, allowing nutrients supply (e.g. Iron). Those modification are probably linked to the interaction with the surrounding mitochondria and endoplasmic reticulum (ER). This unusual interaction seems to play a role in Legionella intracellular replication. This correlation is probably due to the autophagy role of the ER that allows an uptake of nutriment from the cytosol to the vacuole (Swanson et al. 2000).

After attachment to the cell surface, the macrophage will phagocytose the bacteria. It has been reported that L. pneumophila are taken up by macrophages via “coiling phagocytosis” (not all Legionella bacteria, depends on the strain and host cell). The bacteria induce rearrangement of actin filaments to form the coiling asymmetrical pseudopod encircling the extracellular pathogen. Effectors are translocated into the cytosol, powered by ATP hydrolysis and PMF. The dot/icm effector VipA may be an actin nucleator (necessary for actin polymerization) so microfilaments are polymerized for engulfment of the bacteria. The bacteria will be taken up and a nascent phagosome (not yet inherently bactericidal) is formed. (Escoll, et al. 2013)

Other proteins known as cronins (an actin-binding protein) may also be transiently recruited to assist with the phagosome formation. (Escoll et al. 2013)

To start the multiplication process, the conjugation system (Type IV secretion system) gene is necessary for the intracellular growth. Within these loci, 24 essential genes for the host infection are found, for example the following virulence factors: pilE (pilin protein), pilD (prepilin peptidase), which are both important for bacterial growth, mak (macrophage killing) mil (macrophage-specific infectivity loci) or pmi (protozoan macrophage infectivity). (WHO. 2007) Another secretory system found in Legionella is Lvh (Legionella vir homologs), which is also important for intracellular growth, but is not sufficient (the type IV secretory system is still needed even if they have redundant functions).


Avoiding endocytic pathway:

In L. pneumophila, the virulence system, encoded by 26 dot/icm genes (dot: defective in organelle trafficking; icm: intracellular multiplication), involves important factors for host cell entry, intracellular multiplication, and anti-apoptotic host cell signaling. There are also factors to disrupt the phagosome and host cell membranes so the bacteria can enter the extracellular environment for transmission. (Burillo, 2016)

Generally (like in the case for extra-cellular pathogens or apoptotic cells), after phagocytosis, the phagosome fuses with early endosomes (and acquires Rab5, small GTPases). The phagosome continues to mature and become increasingly acidic and obtains lysosome-associated membrane proteins (LAMPs) after interacting with late endosomes. Ultimately the phagosome with late-endosome-like phenotype will fuse with a lysosome to form a phagolysosome where bactericidal killing and digestion occur. Maturation involves lowing the pH in the phagosome via proton transporters called vacuolar H+-ATPases (v-ATPase). (Escoll et al. 2013) This process is disrupted when Legionella are engulfed.

When L. pneumophila get phagocytosed, a neutral pH is maintained in the phagosome for the first six hours. Avoiding early acidification is important for infection. However, at later stages, around 18 hours after uptake, many LCV are acidified and have taken on lysosomal characteristics. Bacteria replicating in macrophages are resistant to low pH environments. (Escoll 2013) Regulating v-ATPase in the macrophage may be significant to avoiding early stage acidification. Outer membrane vesicles (OMVs) shed from L. pneumophila have been reported to block phagosome-lysosome fusion. OMVs are spherical lipid bilayers shed from the outer membrane of gram negative bacteria. OMVs can deliver packages of molecules, including viral proteins. The lipopolysaccharide (LPS) from the OMVs shed by the bacteria may also contribute to arresting phagosome development. (Seeger et al. 2010; Zhan et al. 2015)


Formation of the LCV for Replication:

L. pneumophila is a successful intracellular pathogen because it can successfully replicate inside the cell after engulfment. It does this by manipulating host cell behavior to remodel the phagosome into a replication permissive LCV while concurrently avoiding the usual fusion with a lysosome.

When the bacterium is phagocytosed, it will initially be in a phagosome bound by the former plasma membrane. L. pneumophila orchestrate a process to transform the original endosome membrane of the LCV into an organelle analogous to the rough endoplasmic reticulum to be used for replication. (Escoll et al. 2013; Sherwood and Roy 2016) The dot/icm type IVB secretion system that transports effector proteins across membranes and into host cytoplasm is critical for this process. L. pneumophila transport effector proteins to target host systems involved in vesicle and membrane transport in order to modify the vacuole to take on characteristics of the ER. (see review in Sherwood and Roy 2016; and Tilney et al. 2001) This is explained in more detail below.

During the early stages of infection, the LCV becomes surrounded by smooth vesicles (largely ER-derived) and mitochondria. Very little of the phagosome surface will be exposed to the cytoplasm as organelles (vesicles and mitochondria) will closely associate with and flatten around the phagosome. (Tilney et al. 2001) The LCV will hijack the secretory vesicles exiting the ER and adopt the luminal contents of the vesicles that cycle between the ER and the Golgi apparatus.

It is thought that the secretory vesicles leaving the ER fuse with the LCV. (Escoll et al. 2013) Dot/Icm-translocated bacterial effectors participate in manipulating vesicle and membrane trafficking at this stage. For example, SidM/DrrA and RalF mediate recruitment of the proteins Rab1 and Arf1. Rab1 and Arf1 proteins are usually highly enriched in the cis-Golgi apparatus membrane and facilitate the tethering and fusion of ER-derived vesicles. (Sherwood and Roy, 2016) The vacuole sequestering bacteria also recruits on its membrane the SNARE Sec22b complex, which allows the fusion with ER vesicles. Accordingly, this is thought to enhance fusion of the secretory vesicles with the LCV. Other Dot/Icm translocated substrates then mediate actual recruitment of the ER vesicles to the LCV. (Escoll et al. 2013; Hayley et al. 2010)

(Top) Diagram describing the Rab1 activation pathway in infected macrophages. (Bottom) Fluorescent evidence of the Rab1 presence at the bacteria location.


In addition to recruiting fusion proteins, the bacteria also exploit phosphoinositide lipids (PIs) involved in membrane transport during LCV formation. For example, members of the Sid family of effectors will enhance the levels of PI(4)P on the LCV membrane (similar to the high enrichment of PI(4)P on the Golgi apparatus membrane). (Sherwood and Roy 2016) The enrichment of PI(4)P is likely involved in attachment of ER-derived vesicles to the former plasma-membrane of the LCV. The PIs also provide an important attachment site for effectors on the cytoplasmic face of the LCV. For example, SidC is an effector that uses a PI(4)P binding domain to localize on the cytoplasmic and recruit ER vesicles. (Escoll et al. 2013)

As a result of the hijacking of the ER-derived vesicles, the LCV will acquire proteins usually resident in the ER as well as ER-derived membrane. (Escoll et al. 2013; Tilney et al. 2001) Translocated Dot/Icm effectors also have a role in mitochondria recruitment to the LCV, but less is known of the process and consequences of mitochondrial association to the LCV. Overall, as a result of the first stage of remodeling, the phagosome membrane becomes more like the membrane of the ER-vesicles that are intimately associated to the LCV. (Tilney et al. 2001)

In the next phase the LCV takes on characteristics of the ribosome studded rough endoplasmic reticulum. The ER-derived vesicles and mitochondria surrounding the LCV will decrease and be replaced by ribosomes attached to the LCV membrane. Studies report that the Dot/Icm secretory system is necessary for ribosome recruitment, however, a Dot/Icm-dependent translocated effector mediating ribosomal recruitment has not been described. (Escoll et al. 2013). It is speculated that the initial remodeling of the LCV has rendered it sufficiently ER-like to allow for spontaneous ribosome attraction. In summary, while avoiding endocytic digestion, the bacteria will remodel the LCV by recruiting host ER derived material to form a ribosome-studded LCV. Replication of L.pneumophila commences approximately 4-10 hours after phagocytosis and after establishment of the RER-like former phagosome. (Escoll et al. 2013)

The nutrient environment of the LCV is also important for replication. L. pneumophila recruit ubiquitinated proteins to the LCV mediated by AnkB, another translocated effector. The degradation of the ubiquitinated proteins at the LCV membrane by host proteasomes may provide a source of amino acids for the replicating bacteria. (Escoll et al. 2013) L. pneumophila synthesize amino acid transporters known as “phagosomal transporters” which are necessary for intracellular replication. (reviewed in Hayley et al. 2010) Iron acquisition is also very important for intracellular replication. L. pneumophila have different mechanisms for obtaining iron, including siderophores and transport proteins. They also secrete molecules that can upregulate iron uptake in low-iron conditions. (Zhan et al. 2015)

The L. pneumophila may manipulate the host autophagy system to obtain nutrients. Autophagy is the process where cytoplasmic components and organelles are engulfed by autophagosomes and trafficked to lysosomes for degradation. These macromolecules can be broken down into substrates and recycled for new biosynthesis or energy. It is theorized that autophagosomes may be recruited by the LCV as a source of nutrients for replicating bacteria but studies suggest autophagy contribution is not necessary. (Hayley et al. 2010; Sherwood and Roy 2016)

Once the replication compartment has been established, the Dot/Icm secretion system may not be necessary for replication. However, the Dot-Icm system also transports a number of effectors required for inhibiting host cell apoptosis and egress from the macrophage. (see review in Zhan et al. 2015; and Sherwood and Roy 2016)

Even though the bacterium appears to escape the lysosome-suppressing pathway, the vacuoles in which the bacteria replicate seem to adopt a phagolysosome character. As described before, the late bacteria are more acid-resistant than it previously was, and the acquisition of lysosomal proteins appears necessary for survival and late replication.

Even if the classical endocytic pathway does not apply in the presence of the bacterium, the recruitment of the usual proteins in this pathway (e.g. LAMP-A, cathepsin D, Rab proteins) still takes place, except later than usually. One hypothesis is that eventually, the vacuole will fuse with a lysosomal vacuole, which is a stressful environment but where availability in nutriment is high. Thus, bacterial replication will occur there until the amino acid supply has been fully consumed. (Swanson et al. 2000) (Hayley et al. 2010).

Once the replication compartment has been established, the Dot/Icm secretion system may not be necessary for replication. However, the Dot-Icm system also transports a number of effectors required for inhibiting host cell apoptosis and egress from the macrophage. (see review in Zhan et al. 2015; and Sherwood and Roy 2016)

Then, the spreading begins. The amino acid starvation induces the accumulation of ppGpp, a second messenger that will then trigger the start of the transmission process: cytotoxicity, osmotic resistance, motility, and final evasion from the lysosomal pathway. Regulators of gene expression will influence the transformation from replicative phase bacteria into motile infectious phase bacteria. Cytotoxins production is induced (e.g. legiolysin (hemolytic activity), zinc metalloprotease), what will lead to the host membrane lyse. Such infectious motile bacteria will egress from the host macrophage, often resulting in cell lysis and direct host damage as discussed later in the pathogenesis section.

Secondary Infection

Generally, the initial site of infection for Legionnaire bacteria is the alveolar macrophages. Sometimes legionella bacteria can be introduced via wounds or infected surgical equipment. (Brusch 2016) During Legionnaires disease, after pulmonary infection, bacteria will be present in the blood (bacteremia). (Medical Microbiology, 1996)

Rarely, manifestations of Legionnaire’s disease outside of the lung occurs and these are generally considered secondary to an initial pulmonary infection. Some examples include splenomegaly (enlargement of spleen) and spleen rupture, pericarditis, wounds, joint infection (arthritis), and CNS. (Cunha et al. 2016) Autopsy studies have also described the presence of Legionella in lymph nodes, bone marrow, and kidneys. Secondary infections may be underestimated as physicians are not familiar with extrapulmonary Legionella infection.

Legionella seem to be able to infect extrapulmonary sites via hematogenous spread (distributed by way of the bloodstream) after the pulmonary focus. (Brusch 2016) It is thought that the bacteria can use the infected macrophage as a vehicle for transport throughout the body. When the macrophage lysis, the organisms will be deposited on adjacent tissue making up various organs. (Brusch 2016)

References

Brusch, J.L. (2017). Legionnaire’s Disease: Cardiac Manifestations. Infectious disease clinics of North America, ISSN: 1557-9824, Vol: 31, Issue: 1, Page: 69-80

Cunha, BA, Burillo, A., Bouza, E. (2016) Legionnaires’ disease (Seminar). The Lancet, 387: 376-385https://doi.org/10.1016/S0140-6736(15)60078-2

Escoll, P., Rolando, M., Gomez-Valero, L., Buchrieser, C. (2013) From Amoeba to Macrophages: Exploring the molecular mechanisms of Legionella pneumophila infection in both hosts. In: Hilbi H. (eds) Molecular Mechanisms in Legionella Pathogenesis. Current Topics in Microbiology and Immunology, vol 376. Springer, Berlin, Heidelberg

Hayley J.N. et al. (2010). Molecular Pathogenesis of Infections Caused by Legionella pneumophila. Clin. Microbiol. Rev. 23: 274-298.

Newton, H. et al. (2010) Molecular Pathogenesis of infections caused by Legionella pneumophila. Clin Microbiol Rev. 2010 Apr; 23(2): 274–298. doi:  10.1128/CMR.00052-09

Sherwood, R.K., Roy, C.R. (2016). Autophagy Evasion and Endoplasmic Reticulum Subversion: The Yin and Yang of Legionella Intracellular Infection. Annual Review of Microbiology. 70:1, 413-433

Swanson M.S. and Hammer B.K. 2000. Legionella Pneumophila Pathogenesis: A Fateful Journey from Amoebae to Macrophages. Annual Review of Microbiology 54.

Tilney, et al. (2001). How the parasitic bacterium Legionella pneumophila modifies its phagosome and transforms it into rough ER: implications for conversion of plasma membrane to the ER membrane. Journal of Cell Science 114: 4637-4650

Winn WC Jr. Legionella. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 40. Available from: https://www.ncbi.nlm.nih.gov/books/NBK7619/

World Health organization. 2007. Legionella and the prevention of legionellosis.

Zhan, Xiao-Yong & Hu, Chao-Hui & Zhu, Qing-Yi. (2015). Legionella Pathogenesis and Virulence Factors. Annals of Clinical and Laboratory Research. 3. 15. 10.21767/2386-5180.100015.

Question 4

Bacterial Damage: do the bacteria cause any direct damage to the host (or is the damage fully attributable to the host response, as indicated below) and, if so, what is the nature of the bacterial damage. Can it be linked to any of the signs and symptoms in this case

L. pneumophila can cause both direct and indirect damage to the host after accessing the lung. This bacterium replicates inside alveolar macrophages and can lead to severe pneumonia. Replication inside the host cell occurs for 14 to 16 hours before the progeny exit the host cell. Direct damage to the host via L. pneumophila may result in host cell death as the bacterial infection cycle exhausts the host cell resources. After replication, the bacteria will lyse the host cell and spread to neighboring cells. Iron starvation may be a trigger for bacterial egress. (O’Connor, et al. 2011). The killing of these cells likely occurs via apoptosis and necrosis. During the late stages of replication, L. pneumophila can be found in the cytoplasm, free of the LCV. Escape from the LCV seems to involved pore formation and membrane lysis of the LCV. (Newton, et al. 2010) During the end of replication stage, the bacteria express different genes and phenotypically change into a motile transmissive phenotype. These mature bacteria have shown contact-dependent cell cytotoxicity via small membrane pore development in host cell membranes. (Newton et al. 2010) It is thought that this may assist with bacterial egress from mammalian cells. This would also cause damage to host alveolar cells. Cell lysis is not controlled and can increase damage to neighboring cells. Bacterial factors, including bacterial proteases may also be the cause of tissue damage. This form of bacteria mediated host cell killing is non apoptotic and can occur at low doses of bacteria. The microbial components associated with cell death have yet to be identified, however, flagellin protein may help promote caspase 1-dependent cell death (Isberg, O’Connor and Heidtman, 2009). In addition, bacterial factors including a protease may be responsible for tissue damage present. The primary site of host cell damage includes destruction of pulmonary tissues and cells, including alveolar macrophages.

Fig 5. The mechanisms by which L. pneumophila manipulates host cell death and survival pathways

As shown in the diagram above, the Dot/Icm, described as a type IV-like secretion system allows the bacterial phagosome to evade degradation by the host cell’s endosomal-lysosomal pathway. Furthermore, cell factors such as interleukin-1 may be responsible for signs and symptoms including fever response. This bacterial factor is released from monocytes (Baron S, 1996). The influx of innate immune cells to the site of infection could account for the increase in body temperature as Tom experienced a high fever. Furthermore, other systemic features such as headache are likely due to the immune response to L. pneumophila. In particular, the secretion of tumor necrosis factor may be linked to this symptom. In order for the host to defend against this pathogen, the integrity of physical clearance mechanisms is necessary. Damage to these mechanisms in the immunocompromised, such as Tom who suffers from asthma, make these individuals more susceptible to disease. Complications in immunocompromised populations also include pleural effusion and cavitation (gas-filled area of the lungs in the center of a nodule or a mass or area of consolidation) (Burke, et al. 2016). Untreated Legionnaires disease can lead to more serious complications like respiratory failure, septic shock, and multi-organ dysfunction.

Fig 4. Air spaces are filled with fibrin and inflammatory cells. Gray carbon accumulation around the terminal bronchioles is visible

Indirect damage to the host stems from the host cell’s defence mechanisms against the bacteria. For example, oxygen dependant killing directed by neutrophils of the innate immune system can damage the lung epithelial and endothelial cells. This damage is caused by toxic concentrations of oxygen and leads to the accumulation of protein-rich fluid that floods the alveolar space (Nara et al., 2004). The presence of bacteria, in addition to the death of alveolar macrophages would result in an inflammatory response as neutrophils and monocytes are recruited to the site of infection. Leaky capillaries allow for the influx of serum and increased deposition of fibrin in the alveoli. The resulting pneumonia destroys the air spaces, compromising respiratory functions.


References

Burke, A. C. Burillo, A., Bouza, E. (2016) Legionnaires’ Disease. The Lancet 387: 376-385.

Isberg, R. R., O’Connor, T., & Heidtman, M. (2009). The Legionella pneumophila replication vacuole: making a cozy niche inside host cells. Nature Reviews. Microbiology, 7(1), 13–24. http://doi.org/10.1038/nrmicro1967

Nara C., Tateda, K. Matsumo T., Ohara, A., Miyazaki S., Standiford T.J., Yamaguchi K. (2004). Legionella-induced acute lung injury in the setting of hyperoxia: protective role of tumour necrosis factor-alpha. Medical Microbiology. 53, 727-733. https://doi.org/10.1099/jmm.0.45592-0

Newton, H. et al. (2010) Molecular Pathogenesis of infections caused by Legionella pneumophila. Clin Microbiol Rev. 2010 Apr; 23(2): 274–298. doi: 10.1128/CMR.00052-09

O’Connor, T. et al. (2016) Iron Limitation Triggers Early Egress by the Intracellular Bacterial Pathogen Legionella pneumophila. Infect. Immun. August 2016 vol. 84 no. 8 2185-2197

The Immune Response Questions

Question 1

Host response: what elements of the innate and adaptive (humoral and cellular) immune response are involved in this infection.

Innate Immune Response: The innate immune response is the bodies first line of defense against pathogens and its main priority is to mount a quick and nonspecific immune response. The first component of the innate immune response that the pathogen will come in contact with is the airway epithelium which acts as a physical barrier separating our body from the environment around us. (1) Epithelial cells use pattern recognition receptors (PRRs) such as toll like receptors (TLR) or nucleotide-binding oligomerization domain-like receptors (NLRs) to detect a wide array of pathogen associated membrane proteins (PAMPs) to induce the production of antimicrobial compounds and cytokines to fight the pathogen. (1,3) TLR2 in particular appears to be extremely crucial in the recognition of bacterial peptidoglycans and lipoproteins. (3) In addition, the 2 NLRs ,NAIP4 and IPAF, recognize the flagellin of L. pneumophila and work by restricting bacterial replication in macrophages and epithelial cells. (3) In the case of an infection, epithelial cells depend on nuclear factor-κB to express various kinds of cytokines to attract neutrophils to the site of infection and with the help of CXCL5 and GM-CSF, neutrophil recruitment rates are further increased. (2) When toll like receptors (TLRs) in the epithelial cells signal for the activation of nf-κB, which induce production of various compounds such as proinflammatory cytokines and mucin which work to form a structural barrier to trap small and large particles in the airway. (1) In conjunction with the cilia found on the epithelial cells, the mucus is pushed out of the lungs along with the inhaled particles trapped inside it. (4) Mucus also contains a variety of antimicrobial compounds such as IgA, collectins and defensins to help defend the body against pathogens. (1) Another component of the innate immune response which plays an important role in preventing infection is the presence of resident macrophages which are phagocytic cells which can initiate the inflammatory response and as well as present antigens to B and T cells as part of the adaptive immune response. (1)

Adaptive immune response: Adaptive immunity is a much stronger and sophisticated immune response than the innate immune response. However, the adaptive response is much slower to react due to the fact that it needs to produce antigen specific antibodies which can take several days but the final response can result in long term lasting immunity against the pathogen. (5) As part of the innate immune response, resident macrophages act as antigen presenting cells for B and T cells. (1) Antigen presenting cells trigger the adaptive immunity by activating T and B cells via recognition of major histocompatibility complexes (MHCs) on APCs. Consequently, they generate antigen-specific antibodies in preparation of reinfections (6)

Cytokine production such as interferon gamma-y (INF-y), tumor necrosis-a (TNF-a) and interleukin (IL-6 and IL-1) facilitate inflammatory responses against bacterial infections. For example. TNF-a enhance bactericidal activities of macrophages and inhibits bacterial replication. The importance of these pro-inflammatory cytokines in managing L pneumophila infections is shown in mutated mice that have no IL-1, IL-2, IFN-y and TNF production. As a result, these mice subjects showed a higher susceptibility to L pneumophila infections (7). Upon receiving cytokine signals from macrophages, T cells differentiate into Th1 and Th2 cells. (3) Th1 cells then produce cytokine interferon-γ which plays an important role in controlling growth of intracellular pathogens and increasing production of reactive oxygen and nitrogen species. (3) Reactive Oxygen Species are a group of oxygen radicals such as hydroxyl (OH) and alkoxyl (RO) which have DNA and protein degrading properties to regulate apoptosis. (10) Th1 cells are also capable of recruiting other Th1 cells by expressing CXCL9, CXCL10, and CXCL11 which may help the cell form complex immune responses such as formation of granulomas. (3) Transcription of Th2 cells are controlled by GATA3 and STAT6 and express Interleukin-4, 5, and 13. IL-5 is responsible for regulating eosinophils and IL-4 is critical for B cell proliferation and upregulation of MHC class 2. (3)

When B cells are activated by antigen presenting cells, they begin to form plasma cells which produce antibodies to fight the infection and long lasting memory cells. (9) Antibodies work by blocking host cell and viral interactions and acting as markers so macrophages can more easily identify them.

Infants on the other hand rely heavily on breast milk from their mother for passive immunity against pneumonia. (8) In a study looking at the morbidity and mortality of infants with pneumonia, they found that infants who were not adequately breastfed in their first 23 months showed higher rates of morbidity and mortality. (8)

References:

(1) Eisele NA, Anderson DM. Host Defense and the Airway Epithelium: Frontline Responses That Protect against Bacterial Invasion and Pneumonia. Journal of Pathogens 2011;2011:e249802.

(2) Yamamoto K, Ahyi AN, Pepper-Cunningham ZA, Ferrari JD, Wilson AA, Jones MR, et al. Roles of lung epithelium in neutrophil recruitment during pneumococcal pneumonia. Am J Respir Cell Mol Biol 2014 Feb;50(2):253-262.

(3) Eddens T, Kolls JK. Host defenses against bacterial lower respiratory tract infection. Current opinion in immunology 2012 Aug;24(4):424-430.

(4) Mall MA. Role of cilia, mucus, and airway surface liquid in mucociliary dysfunction: lessons from mouse models. J Aerosol Med Pulm Drug Deliv 2008 Mar;21(1):13-24.

(5) Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P. The Adaptive Immune System. 2002.

(6) Massiss L, Zamboni D. Innate Immunity to Legionella Pneumophila. Front Microbiol. 2011; 2(109)

(7) Astrat S, Dugan A, Isberg R. The Frustrated Host Response to Legionella pneumophila Is Bypassed by MyD88-Dependent Translation of Pro-inflammatory Cytokines. PLOS pathogens. 2014

(8) Lamberti LM, Zakarija-Grković I, Fischer Walker CL, Theodoratou E, Nair H, Campbell H, et al. Breastfeeding for reducing the risk of pneumonia morbidity and mortality in children under two: a systematic literature review and meta-analysis. BMC public health 2013;13 Suppl 3(Suppl 3):S18.

(9) Medical Microbiology. 4th ed. Galveston (TX): University of Texas Medical Branch at Galveston; 1996.

(10) H.-U Simon, A Haj-Yehia and F Levi-Schaffer. Role of reactive oxygen species (ROS) in apoptosis induction. APOPTOSIS. November 2000; 5[5]: 415-418.

Question 2

Host damage: what damage ensues to the host from the immune response?

Cytokines from the early phase of the innate immune response are protective to the host at normal levels. However, extremely elevated cytokine concentrations can become fatal to the host as it induces sepsis and potentially toxic-like death (Hoffman, 2007). TNFa and IL-1 have been identified as central mediators of septic shock and are produced in high concentrations due to excessive stimulation of host immune cells with LPS (Shapira et. al., 1996). This large production of pro-inflammatory cytokines contributes to dysfunctions in coagulation and eventual hypotension, which could manifest into acute respiratory distress syndrome (Stearns-Kurosawa et. al., 2011)

CD4+ and CD8+ T cells activated by dendritic cells cause localized tissue damage during the adaptive immune response (Park, 2016). Cell death of macrophages infected with L. pneumophila often occur in order to eliminate the spread of this pathogen. Macrophage pyroptosis, a form of apoptosis that is caspase-1 dependent, begins with the activation of the NLRC4 inflammasome. This complex induces the fusion of the infected macrophage with the phagolysosomes, resulting in cell death. Thus, as L. pneumophila infection progresses, the number of alveolar macrophages in the host decreases since the body attempts to eliminate the pathogen (Brown, 2016).

With injury or infection, the rise in inflammatory cells may be sufficient enough to damage the endothelial layer and increase its permeability (which inflammatory responses are effective in doing). As a result, fluid builds up in the interstitial and alveolar spaces due to disruption of pressures - pulmonary edema (Murray, 2011). This accumulation of edematous fluid restricts proper gas exchange and may result in dyspnea (shortness of breath). L. pneumophila can cause massive consolidation and necrosis of lung parenchyma and is associated with a high mortality rate if the infection is not treated (Damjanov, 1996).

As Legionnaire’s Disease is often confused with and misdiagnosed as other forms of pneumonia, more research is required to gain a better understanding of the host damage inflicted by the immune response to L. pneumophila infection.


Brown, A. S., Yang, C., Hartland, E. L., & Driel, I. R. (2017;2016;). The regulation of acute immune responses to the bacterial lung pathogen legionella pneumophila. Journal of Leukocyte Biology, 101(4), 875-886. 10.1189/jlb.4MR0816-340R


Damjanov, I. (1996). Pathology for the health-related professions. Philadelphia: W.B. Saunders Company.

Hoffman, P. S., Friedman, H., Bendinelli, M., & SpringerLink ebooks - Biomedical and Life Sciences. (2007;2008;). Legionella pneumophila: Pathogenesis and immunity. London;New York;: Springer.10.1007/978-0-387-70896-6

Murray, J. F. (2011). Pulmonary edema: Pathophysiology and diagnosis. The International Journal of Tuberculosis and Lung Disease : The Official Journal of the International Union Against Tuberculosis and Lung Disease, 15(2), 155.

Park, B., Park, G., Kim, J., Lim, S. A., & Lee, K. M. (2017). Innate immunity against Legionella pneumophila during pulmonary infections in mice. Archives of pharmacal research, 40(2), 131-145.

Shapira, L., Soskolne, W. A., Houri, Y., Barak, V., Halabi, A., & Stabholz, A. (1996). Protection against endotoxic shock and lipopolysaccharide-induced local inflammation by tetracycline: correlation with inhibition of cytokine secretion. Infection and Immunity, 64(3), 825-828.

Stearns-Kurosawa, D. J., Osuchowski, M. F., Valentine, C., Kurosawa, S., & Remick, D. G. (2011). The Pathogenesis of Sepsis. Annual Review of Pathology, 6, 19–48. http://doi.org/10.1146/annurev-pathol-011110-130327



Question 3

Bacterial evasion: how does the bacteria attempt to evade these host response elements.

Properties of the Cell Envelope

Legionella bacteria possess many common bacterial structures on its cell surface, including lipopolysaccharide (LPS), outer-membrane vesicles (OMVs) flagella and type IV pili (1). The OMVs are an important factor in the early stages of infection because it facilitates the binding and transfer and fusion of the bacterial to host cell surfaces so that it can engulfed by the host cell. However, as an intracellular pathogen, Legionella bacteria must find a method to avoid being degraded after being phagocytosed. It has been shown that once the bacteria are phagocytosed, it is capable of modifying the pH of its phagocytic vacuole, and the OMVs prevents the fusion of the phagosome with lysosome; the highly acidic environment is not created, and the bacteria is able to avoid the degradation process (2). The type IV also contributes in the attachment and entry of the bacteria into target host cells, however, the most important function of this cell envelope structure is its ability to promote biofilm development and adhesion. This promotes Legionella adherence and aggregation in biofilms, and the remodeling of biofilm structure via twitching motility helps to facilitate the bacteria’s survival in a variety of physiological conditions (3).

The cellular surfaces of bacteria present many antigenic targets, which prove to cause difficulty for bacterial pathogens in hiding their complex surface of proteins and carbohydrates from the host’s immune system and Toll-Like receptor (TLR) recognition while trying to expose important molecules such as adhesins and invasins (4). A common mechanism for disguising bacterial surfaces is the expression of a carbohydrate capsule (4). This mechanism is commonly used by extracellular bacterial pathogens which systematically circulate in the body and these pathogens that express surface capsules usually also have filamentous adhesins such as fimbriae and pili that protrude through their capsular surface and enables the adhesins to bind to host receptors while keeping the bacterial surface hidden from host immunity (4). Lipopolysaccharide (LPS) is one of the main surface-exposed component of Gram-negative bacteria and an important molecule in both the bacteria and the host A common mechanism for disguising bacterial surfaces is the expression of a carbohydrate capsule (4). The main component of LPS, lipid A, is highly conserved in most Gram-negative bacteria and plays a key role in activating TLRs such as TLR4, however, the outer part of LPS is composed of highly variable carbohydrates, which gives each strain a particular serotype (O antigen) (4). This explains how different strains of the same species can re-infect the same host due solely on O antigen differences (4). Gram-negative bacteria have developed secretion systems to export virulence factors across bacterial membranes and into the supernatant or directly into host cells (4). With regards to virulence factor secretion of toxins and immune modulators, Gram-negative bacteria involve both type III secretion systems (T3SS) and type IV secretion systems (T4SS), which can insert various molecules into host cells (4). These two systems can secrete effector molecules such as toxins that can kill host cells, mediate bacterial uptake or invasion, secrete effectors that re-program vesicular transport to enhance intracellular parasitism, mechanisms to paralyze phagocytosis, and effectors that can alter immune functions to enhance evasion (4).

The Dot/Icm transporter; Source: (8)

Survival Inside the Host Cell

To replicate inside a host cell, the Legionella bacteria must prevent the activation of the infected cell so that it can be able to multiply inside the cell. This is facilitated by Mip protein encoded by the bacteria which mimics the structure and function of the proteins of the eukaryotic protein family FK506 and inhibits the activation of the infected cell (5). By doing so, this helps Legionella survive within the host cell and blocks the killing of these host cells.

Growth of Legionella inside an alveolar macrophage using LCV; Source: (10)

In addition, Legionella bacterial uses a Dot/lcm type IV secretion system to invade the host cell and create signal molecules, to facilitate the growth of the pathogen. When the phagocytosis is initiated by the alveolar macrophage, the bacteria will release bacterial effectors proteins to the cytosol. This results in the creation of a vesicle that is similar to one of the endoplasmic reticulum known as the Legionella-containing vacuole (LCV). The LCV prevents the recruitment of immune cells to the infected cell, providing the bacteria a safe environment to replicate. (6). Moreover, the type IV secretion system promote intracellular replication of the bacteria, it also inhibits host cell apoptosis and facilitates the release of the bacteria.

The intracellular bacteria, L. pneumophila, can avoid phagolysosome fusion and replicate within alveolar macrophages. This is done through an intracellular mechanism that inhibits of vacuolar-ATPases. With this mechanism, acidification of the LCV will not occur, causing the lysosomal enzymes to not get activated, and thus the Legionella can still continue to divide and thrive within the cell (9).

Killing Alveolar Macrophages

If the bacteria succeed in infecting the alveolar macrophage, it causes the host cell to undergo lysis, killing the macrophage while releasing a high amount of the replicated bacteria (7). By doing so not only is the bacteria able to multiply in numbers, it also decreases the number of alveolar macrophages present in the lungs, and thus decreases the immune response to subsequent Legionella bacteria entering the airways.

References:

1. Shevchuk, O., Jäger, J., & Steinert, M. (2011, April 25). Virulence properties of the legionella pneumophila cell envelope. Retrieved March 03, 2018, from https://www.ncbi.nlm.nih.gov/pubmed/21747794

2. Fernandez-Moreira, E., Helbig, J. H., & Swanson, M. S. (2006, June). Membrane vesicles shed by Legionella pneumophila inhibit fusion of phagosomes with lysosomes. Retrieved March 03, 2018, from https://www.ncbi.nlm.nih.gov/pubmed/16714556

3. Coil, D. A., & Anné, J. (2009, April). Twitching motility in Legionella pneumophila. Retrieved March 03, 2018, from https://www.ncbi.nlm.nih.gov/pubmed/19243440

4. Finlay, B. B., & Mcfadden, G. (2006). Anti-Immunology: Evasion of the Host Immune System by Bacterial and Viral Pathogens. Cell, 124(4), 767-782. doi:10.1016/j.cell.2006.01.034

5. Riboldi-Tunnicliffe, A., König, B., Jessen, S., Weiss, M. S., Rahfeld, J., Hacker, J., . . . Hilgenfeld, R. (2001, September). Crystal structure of Mip, a prolylisomerase from Legionella pneumophila. Retrieved March 03, 2018, from https://www.ncbi.nlm.nih.gov/pubmed/11524681

6. Luo, Z. Q., & Isberg, R. R. (2004, January 20). Multiple substrates of the Legionella pneumophila Dot/Icm system identified by interbacterial protein transfer. Retrieved March 03, 2018, from https://www.ncbi.nlm.nih.gov/pubmed/14715899

7. Losick, V. P., & Isberg, R. R. (2006, September 04). NF-kappaB translocation prevents host cell death after low-dose challenge by Legionella pneumophila. Retrieved March 03, 2018, from https://www.ncbi.nlm.nih.gov/pubmed/16940169

8. Roy CR, Jonathan CK. 1970. Evasion of Phagosome Lysosome Fusion and Establishment of a Replicative Organelle by the Intracellular Pathogen Legionella pneumophila. Madame Curie Bioscience Database [Internet]. U.S. National Library of Medicine.

9. Xu, L., Shen, X., Bryan, A., Banga, S., Swanson, M. S., & Luo, Z. (n.d.). Inhibition of Host Vacuolar H -ATPase Activity by a Legionella pneumophila Effector. Retrieved March 10, 2018, from http://journals.plos.org/plospathogens/article?id=10.1371%2Fjournal.ppat.1000822

10. (n.d.). Retrieved March 3, 2018, from 18. https://www.researchgate.net/figure/Compartmentalization-of-the-metabolism-of-L-pneumophila-Within-eukaryotic-host-cells-L_fig1_266086285

Question 4

Outcome: is the bacteria completely removed, does the patient recover fully and is there immunity to future infections from this particular bacteria ?

Patient Outcome


File:National-incidence.png
Incidence of Legionella pneumophila infection between 2000-2015, in the United States.

Humans may become infected with Legionella pneumophila via contaminated aerosol inhalation, delivering the pathogen to the lung epithelial cells, where it is able to infect alveolar macrophages, inside which it replicates [3]. Upon infection with the pathogen, the adaptive immune system develops a strong B cell response specific to the pathogen, which is observed to occur more rapidly following secondary infection, as do most secondary infections after the development of immune memory towards a pathogen [3]. Further, experimental immunization with polyclonal immunoglobulins has been shown to provide protection from subsequent introduction or infection with L. pneumophila [3]. Interestingly, lipopolysaccharide targeted immunoglobulin opsonization of Legionella pneumophila in mice subjects showed a decrease in growth of the pathogen in the lungs [1], so memory B cells producing LPS antibodies would provide some protection during reinfection; cell-mediated immunity also plays a crucial role during primary infection of the host [6]. Though Legionnaires disease can prove deadly, if treatment is received in time approximately 90% of those infected make a full recovery [2]. The observed mortality rate associated with Legionella pneumophila among immunocompromised patients is approximately 2-25% [4]. Following treatment with appropriate antibiotics, the bacteria is completely removed from the patient’s body. CD4+ T-helper 2 cells produce Interleukins 4-6 and activate B-lymphocytes, driving them to differentiate into plasma cells which synthesis antibodies towards the pathogen. Since B cells differentiate into antibody producing plasma cells, some of these cells further specialize into long-lived plasma cells or B-memory cells, so if the patient is re-infected in the future there will be some immunity towards the pathogen. Further, interferon-gamma produced by T cells aids in the removal of the pathogen from the body [5]. Overall, while the patient is expected to make a full recovery following elimination of the pathogen from the body, unfortunately any scarring of lung tissue resultant from infection will likely not fully recover.


1. Brieland, J. K., Heath, L. A., Huffnagle, G. B., Remick, D. G., McClain, M. S., Hurley, M. C., Kunkel, R. K. et al. (1996). Humoral immunity and regulation of intrapulmonary growth of Legionella pneumophila in the immunocompetent host. J. Immunol. 157: 5002–5008. 2. Dooling KL, Toews KA, Hicks LA, et al. (2015). Active Bacterial Core Surveillance for Legionellosis–United States, 2011–2013. MMWR Morb Mortal Wkly Rep. 64(42):1190–3 3. Joller, N., Spörri, R., Hilbi, H., & Oxenius, A. (2007). Induction and protective role of antibodies in Legionella pneumophila infection. European journal of immunology, 37(12), 3414-3423. 4. Mykietiuk, A. Carratala, J. Fernandez-Sabe, N. Dorca, J. Verdaguer, R. Manresa, F. Gudiol, F. (2005). Clinical Outcomes for Hospitalized Patients with Legionella Pneumonia in the Antigenuria Era: The Influence of Levofloxacin Therapy. Clinical Infectious Diseases. 40(6): 794-799. https://doi.org/10.1086/428059 5. Salins, S., Newton, C., Widen, R., Klein, T. W., & Friedman, H. (2001). Differential Induction of Gamma Interferon in Legionella pneumophila- Infected Macrophages from BALB/c and A/J Mice. Infection and Immunity, 69(6), 3605–3610. http://doi.org/10.1128/IAI.69.6.3605-3610.2001 6. Susa, M., Ticac, B., Rukavina, T., Doric, M. and Marre, R. (1998). Legionella pneumophila infection in intratracheally inoculated T cell-depleted or -nondepleted A/J mice. J. Immunol. 160: 316–321.