7.+Case+study+in+full

=Case study in full:= An infectious agent is a pathogen which can cause disease. Legionnaires disease is caused by the infectious agent legionella pnemophila.

Legionnella pneumophila is of the genus Legionella from the family Legionellaceae. Figure 1, shows a scanning electron micrograph of the infectious agent.There are many different species of Legionella with 53 species currently discovered. 70 serogroups of Legionella have been identified (Guyard 2011) with //L. pneumophila// being the primary human pathogenic bacterium in this group. //L. pneumophila// serogroup 1 alone is responsible for 95% of all cases of Legionnaires disease. (Valero.2009)

L. pneumophila, pictured in figure 2, are flagellated, non-capsulated, thin bacterium. They are aerobic, non-spore forming, gram negative cocco bacilli. The organism is approximately 2µm - 20µm in length and 0.3-0.9µm in width. The cell envelope is composed of branched-chain fatty acids and distinctive ubiquinones, whose structural differences have been used to classify different Legionella species. In infected tissue and clinical specimens, the organisms are coccobacillary and often measure 1–2 μm.

The optimum growth temperature for the bacteria is 35 degrees Celsius. At low temperatures the bacterium enters a dormant stage until exposed to more favourable conditions. L.pneumophila thrives in areas where there are high concentrations of rust, algae, and organic particles. This supplies the bacteria with a source of iron and nitrogen necessary for providing nutrients for growth, as well as protection from harsh chemicals.

Legionella Pneumophila are normally found in non- marine aquatic environments such as lakes and ponds. They act as intracellular pathogens of amoebae and in biofilms. Biofilms are highly efficient and stable ecosystems where resident L.pneumophila are well adapted to survive prolonged periods of environmental stress. L. pneumophila are spread primarily by water, but can colonize in any warm, damp environments and manmade water systems such as air conditioning and hot water systems. (Guyard.2011)

There is a parasitic relationship between Legionella and some of its protozoan hosts like Hartmanella vermiformi and Acanthamoeba Castellanies. The bacterium is able to invade and then multiply in the host. Growth of L. pneumophila in certain protozoan such as Acanthamoeba castellanii enhances the invasion of epithelial cell macrophage in humans. A cycle of replication within amoebic hosts increases the virulence in humans improving their ability to replicate inside eukaryotic cells such as macrophages, epithelial and endothelial cells. (Declerck.2007)

Humans become infected with the bacterium by inhaling aerosols from the contaminated water sources. L. pneumophila bacterium causes disease in humans by replicating inside alveolar macrophages. Virulent forms of the L. pneumophila bacterium have an ability to prevent phagosome to lysosome fusion, resulting in severe pneumonia known as legionnaires disease. Non virulent strains of the L. pneumophila species are unable to cause disease as they are defective in replicating inside the host cell. Figure 3, shows how the bacterium enters a human host and replicates to cause disease.



The virulence of the l. pneumophila, depends on its species specific genes. Salt sensitivity, motility and the presence of contact dependent cytotoxicity have an ability to affect the function of the dot/icm system. The inactivation of the defective for organelle trafficking/intracellular multiplication (dof/icm) gene results in the inhibition of the phagosome- lysosome fusion preventing intracellular growth in a host. (Vogel.1999)

Legionella are gram negative bacilli which do not grow on routine media used in Bacteriology. It requires complex nutritional requirements such as high cysteine levels, iron and low sodium levels to grow. As a result it is often difficult to isolate and identify outside of the host.

Pathogenecity is described as the ability of a parasite to inflict damage on the host target. Legionella bacteria are an intracellular organism found mainly in watery areas. Legionella is a gram negative bacteria that spreads through water vapour which when inhaled by a human can lead to the progression of the disease. Legionella can either lead an independent existence or live as a parasite. The bacteria kill mammalian cells in two phases: by the mediation of cell death and injury during the early stages of infection, followed by an independent and intensified cell death during later stages of the infection, stimulated by a pore-forming activity. Once Legionella enter the host cell the bacteria are known to be able to avoid endosomal and lysosomal activity, however the immune response is quick to react recruiting macrophages to the area of infection due to the expression of the cytokine IL-8 (Interlukin-8) induced by the Legionella bacteria flagellum .” ** //Moreover,// ** // **Legionella ** //** //flagellin seemed to be involved in IL-8 expression, since a// ** // **flaA ** //** //knockout mutant, defective in flagellin production, failed to induce IL-8 mRNA immediately after the infection//”. ** ** __Mechanisms of Legionella pneumophila-induced interleukin-8 expression in human lung epithelial cells.__ ** ** __Hiromitsu Teruya et al.__ ** After the expression of il8 the recruitment of the alveolar macrophages induces them to take up the Legionella by phagocytosis. After the Legionella is phagocytosized they undergo a physiological transformation which integrates the Legionella bacteria into part of the phagosomes. The Legionella that have control of the phagosome are now able to freely initiate a secretion system encoded by the Dot ICM gene. The secretion system includes signalling proteins/effectors which result in averting the overall maturation of the phagosome. One of the signalling proteins expressed by the Legionella is known as cyclic diguanylate, is the regulator of a large number of variably important processes in the Legionella bacteria. These processes include. Motility the independent movement of the Legionella bacteria Bio film formation helps the Legionella bacteria exist independent of a host Cell division the Legionellas ability to proliferate Virulence and Growth. The //Legionella bacteria also expresses an// effector protein effector proteins secreted by the DOT ICM known as DrrA. This effector protein acts as an exchange factor for guanine nucleotide (GEF) present in Rab1 ( Rab1 being a GTPase that regulates the transport of endoplasmic-reticulum-derived vesicles), therefore activating this GTPase causes the aggregation of these vesicles to the area of expression. DrrA maintains the active state of Rab1 by a process called Ampylation. This is done by the addition of an adenosine 5'-monophosphate (AMP) to Tyr77 modification that happens after translation mechanism. The small Guanosine Triphosphatase (GTPase) Rab1 is the target for the phagosomes containing Legionella. This targeting results in the recruitment of endoplasmic reticulum derived vesicles. The vesicles surround the Legionella and flatten it until the phagosome fuses with vesicles. Ribosomes are later recruited to the phagosome containing Legionella Once a Legionella is in an endoplasmic derived vesicle it is hidden from the lysosomal activity Now the Legionella cannot be attacked by the lysosomes they multiply freely within the macrophage They grow in the presence of L-cysteine and iron growing flagella becoming mobile and then try to exit the macrophage by cycles of phagocytosis and multiplication



Optimal growth temperature is 28-40°C; organisms are dormant below 20°C and are killed at temperatures above 60°C.

Legionnaire’s disease is caused by bacteria and it’s a form of pneumonia. The name Legionnaire’s disease came from people who attended the American legion’s convection in Philadelphia in 1976 where they got affected by the disease. Legionella are mostly found in cooling waters, humidifiers and potable water distribution systems. This disease can be spread by erosolisation or aspiration of water contaminated with legionella organism.

The epidemic of this disease is the consequence of the rapid growth of bacteria, and leads to the rose of artificial sources from the failure of controlling and prevention measures. In England and Wales about 40% of cases were reported and it due to foreign travel. //(Edwards, M et al 2008)//

According to the “Bureau of Communicable Disease Control” it been reported that in the USA there were 8,000 to 18,000 cases of the disease reported each year. ( //McNabb // //SJ. Et al 2005) // . In 1995 – 2005 more than 32,000 of Legionnaires’ disease were reported and over 600 outbreaks were also reported to the European Working Group for legionella infections.

This disease is more likely to be increased as the population becomes more elderly, and there will be a shortage of data in relation to legionella in developing countries, most legionella disease is misdiagnosed worldwide, however diagnosis and surveillance can be improved in developing countries and a better result would be expected in order to reveal far higher level of morbidity and mortality than are currently recognised, this will also improve the awareness about the incidence and how it is spread.

In the south of Wales, //(September 2010)// there is a new outbreak of legionnaire’s disease which killed about three people. Some people who have been ill never knew they got the disease till an investigation took place in certain area of South Wales, which identify a total of 19 people who suffer from the disease//. (bbc news one Wales)//

In Europe, 5,960 cases were reported in 2008 from 34 European countries, but the highest incidence was from France (1,244), Spain (1,219) and Italy (1,107). However, the rates per million population were highest in Switzerland (28.6), Slovenia (23.7), Denmark (23.3) and the Netherlands (20.5). On the other hand, in 2009, 808 cases of travel-associated Legionnaires’ disease were reported from European countries.

In Australia, especially Melbourne in year 2000 there were an outbreak of the disease were 125 cases were verified cases of legionnaires pneumonophia, but only 95 was hospitalised, and about 4 died from the outbreak. The cause was due to infection to the cooling tower at their newly open aquarium, but in 2001 the government introduced a controlling system for the infection.

In December 2011, the Hong Kong’s headquarters were contaminated with the bacterial that caused Legionnaires’ disease. A minister was hospitalised with the potentially fatal disease. There were traces of the bacteria which are 14 times above the acceptable level. The building has been disinfected by authorities, and the Hong Kong’s health centre protection has been gathering samples from various sites.

[[image:group-b-omed0074/Legionnaires_disease_cases_1988_-_2007.png caption="Legionnaires_disease_cases_1988_-_2007.png"]]

 * This graph is a statistics that showed reported cases over the past 20 years, an average of 200-300 cases every year. ( **// nemco-utilities.co.uk) //

Legionnaire’s disease is a non-contagious disease caused by the bacteria found in water systems. This disease is mostly caused by Legionella pneumophilia which mostly occur all through winter months. Moreover, the symptoms of this disease are often variable (Kirrage et al, 2003) Hence, there are various symptoms of Legionnaire’s disease and high fever is the earliest signs of the illness in which the disease appears 2 to 10days after it has been exposed to the bacteria causing the disease. Fig1 : Onset dates of Symptoms of Legionella infection of patients in wales from July 2010 to September 2010 ( Kemarou. M and Evans .M.R , 2010). The figure above indicated that the onset dates of symptoms was ranged from 4th of august to september 2010 in which none of the patients in the community was known to have travelled as at that time. Other main symptoms of the Legionnaires disease are Loss of energy, coughing of blood, gastrointestinal symptoms as such as diarrhoea, nausea, vomiting and abdominal pain (Magnussen.M,2004), general discomfort, shortness of breathe, Loss of energy, shaking chills, joint pain, muscle aches, stiffness and chest pain (Swartz.M,1979). The high fever is due to the bacteria that caused the illness in which the time at which the patient is exposed to bacteria for this illness is shorter and is few hours to 2days compare to the time at which the patient is exposed to bacteria and beginning of the illness which is about 2 to 10days. The headache and muscle pain is due to symptoms of Pontiac fever, but because of the doubts of the definition and pathogenesis of Pontiac fever people with mild legionnaires are also suspected to have Pontiac fever. Shortness of breath in patient with legionnaire’s disease is due to lungs problem because the bacteria spreads to the chest and tighten the chest which causes shortness of breath. Loss of energy may be due to depression and tiredness which weakens the patient and causes total discomfort while cough is due to the lungs infection and is one of the main distinctive symptoms <span style="background-color: #ffffff; display: block; font-family: cambria,serif; font-size: 16px; text-align: justify;">(Guayard et al, 2011). <span style="background-color: #ffffff; display: block; font-family: cambria,serif; font-size: 16px; text-align: justify;"> Legionnaires Disease (LD) was in the past associated with a high level of mortality (50% of all hospitalised cases if treatment started late) but now due to earlier detection as well as a better understanding of the drugs needed to treat Legionnaires disease these rates have decreased. Antimicrobials are used as an effective treatment for LD and are split into different groups – Macrolides, Quinolones, Tetracyclines and Rifampicin. This aids the immune system in fighting off the bacteria and allows the patient to recover. Antimicrobials are given to the patient upon admittance intravenously. The choice of microbial agent is dependent on the efficacy and cost of the drug to be used as well as condition of the patient. This takes into account any other medical problems the patient may be suffering from such as being immunosuppressed or if the patient is pregnant. Treatment is also dependent on the severity of a suspected case of Legionnaires disease and in the most severe cases patients are treated with non-specific antimicrobials containing antimicrobials that are effective against LD whilst being tested for the strain. The patient is then switched to the more effective treatment upon diagnosis. Alongside the chosen treatment option, patients receive IV fluids to maintain hydration and in severe cases can also receive oxygen or assisted breathing.

The most common antimicrobial treatment is Macrolides which inhibit protein synthesis at the 50S ribosomal complex and causes the growth of Legionnella pneumophilia to be inhibited through decreased translocation (sevilla-sanchez et al, 2010). Erythromycin was the first Macrolide produced and is commonly used as a first line treatment. This is due to its low dosage and effective efficacy. Although newer drugs such as Azithromycin and Clarithromycin have proven to be equally effective if not more effective than erythromycin but at lower doses (Zahar et al, 2008). As you can see in figure 1 (below) the low dosages of macrolides mean a lower toxicity to the body which is good treatment option during pregnancy and also in immunosuppressed patients.



Figure 1 – A table showing doses of drugs and route drug is given in patients suffering from legionnaires disease (Diederen. B.M.W, 2008).

Other treatment options include Tetracyclines which also target the 50S ribosomal complex and Quinolones which inhibit DNA synthesis which are observed in figure 2 (below). They are used to a great extent as a secondary treatment option in patients suffering from LD. Rifampicin targets RNA synthesis (figure 1) but due to the variation of efficacy its effectiveness has been questioned (Retsama et al, 2001). This variation has caused Rifampicin to not be used as a treatment option in the first instance but can be used as a last resort. Combined treatment alongside erythromycin was also looked into but showed that erythromycin alone had increased efficacy and so combined treatment using Rifampicin is also not recommended (Diederen. B.M.W, 2008).



Figure 2 – A table showing the mechanism of action for the main antibacterial classes (Retsama et al, 2001).

A new therapy that is being currently looked into are antimicrobial peptides (AMP’s) which are natural antibiotics that target the fatty acid composition of the phospholipid membrane. Warnericin RK is already showing promising results in the treatment of LD (Verdon et al, 2010). AMP’s will provide an alternative treatment option for LD and will allow a treatment to still be in place in the event of resistance to other antimicrobial drugs such as erythromycin.

**__Group conclusion:__** As a group we were provided with a gram stain showing gram negative bacteria and also an image of the bacteria growing on buffered charcoal yeast extract. Along with the patient symptoms of fever, malaise and especially respiratory problems, the patient was diagnosed with legionnaire’s disease caused by legionella pneumophila. Legionnaire Pneumophila is spread through contact with contaminated water vapour,enters the host cell and avoids detection. Then the immune system through IL-8 recruits macrophages which take up legionella through phagocytosis. This integrates into the phagosome and starts secreting signalling proteins/effectors blocking maturation of the phagosome. Vesicles migrate to the area where GTPase is expressed surrounding legionella and fuse with the phagosome. Ribosomes containing legionella are recruited around the phagosome allowing legionella to be hidden from the immune system and proliferation occurs. It lyses the host cell causing cell death and destruction upon release. It is most commonly contracted in middle aged to elderly patients in developed countries. Various symptoms are caused by legionnaires disease such as fever, vomiting, shortness of breath along with muscle pains which present 2- 10 days after exposure to L.Pneumophila. Due to the severity of the patients symptoms leading to multi-system failure, antimicrobials should be administered intravenously and assisted breathing or oxygen should be used to aid recovery. Using a cocktail of drugs should be used to block resistance from occurring. Following treatment the patient should have no lasting effects and will recover fully.

=__**References:**__=


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<span style="background-color: #ffffff; font-family: 'Times New Roman',serif; font-size: 12pt;">Posted on Thursday 16th September 2010
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