Pseudomonas and Legionella

1. Pseudomonas and Legionella • Pin Lin (凌 斌), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw • References: 1. Chapter 34 & 38 in Medical Microbiology (Murray, P. R. et al; 5th edition) 2. 醫用微生物學 (王聖予 等編譯, 4th edition)

2. Outline • Physiology & Structure • Pathogenesis & Immunity • Lab Diagnosis, Treatment, & Prevention

3. Pseudomonas (pseudes, false; monas, a unit or monas) Structure and Physiology 1. Gram-negative rods w/ polar flagella 2. Non-fermentative 3. Obligate aerobe, using carbohydrates via respiratory mechanism (O2 => Electron receptor). 4. Sometimes grow anaerobically (Nitrate or Arginine => Electron receptor). 5. Cytochrome Oxidase -positive (=> diagnosis) 6. Pigments (some strains)

4. Pseudomonas and related organisms Consists of ~ 10 species Pseudomonas aeruginosa: most common, opportunistic infections of multiple sites Burkholderia cepacia: RT infection in cystic fibrosis patients, UTI, opportunistic infections B. pseudomallei: sepsis or chronic pulmonary infection Stenotrophomonas maltophilia: opportunistic infections Acinetobacter baumannii:opportunistic infections of RT Moraxella catarrhalis: RT infections

5. P. Aeruginosa (refer to green pigment) Forms round colonies with a fluorescent greenish color, sweet odor, and b-hemolysis. Pyocyanin- nonfluorescent bluish pigment; pyoverdin- fluorescent greenish pigment; pyorubin, andpyomelanin Some strains have a polysaccharide capsule. Identification of P. aeruginosa is usually based on colonial morphology (flat colonies w/ spreading border), b-hemolysis, oxidase positive, the presence of characteristic pigments and sweet odor, and growth at 42 oC.

6. Outline • Physiology & Structure • Pathogenesis & Immunity • Lab Diagnosis, Treatment, & Prevention

7. P. aeruginosa Pathogenesis and Immunity 1. This organism is widely distributed in nature and is commonly present in moist environments in hospitals. It is pathogenic only when introduced into areas devoid of normal defenses, e.g., 1. Disruption of mucous membrane and skin. 2. Usage of intravenous or urinary catheters. 3. Neutropenia (as in cancer therapy). 2. P. aeruginosa can infect almost any external site or organ. 3. P. aeruginosa is invasive and toxigenic. It attaches to and colonizes the mucous membrane or skin, invade locally, and produces systemic diseases and septicemia. 4. P. aeruginosa is resistant to many antibiotics. It becomes dominant when more susceptible bacteria of the normal flora are suppressed.

8. Virulence Factors of P. aeruginosa-I Structural Components 1. Pili and nonpilus adhesins. 2. Polysaccharide capsules (seen in cultures from patients with cystic fibrosis): protects from antibiotics (aminoglycosides) killing and phagocytosis. 3. LPS: endotoxin, multiple immunotypes. 4. Pyocyanin: impairs ciliary function;induce IL-8; catalyzes production of toxic forms of oxygen that cause tissue damage.

9. Virulence Factors of P. aeruginosa-II Toxins & Enzymes Elastases: LasA (serine protease) & LasB (zinc metalloprotease); destruct elastin-containing tissues and the lung parenchymal damage and hemorrhagic lesions (Ecthyma Gangrenosum); help bacteria spread and inhibit neutrophil chemotaxis; induces antibodies in chronic infections. Hemolysins: Phospholipase C (Heat-sensitve): breaks down lipid & lecithin Rhamnolipid (Heat-stable): inhibits ciliary activity. Exotoxin A: blocks protein synthesis; immunosuppressive; causes dermal tissue necrosis. Exoenzyme S and T: ADP-ribosyltransferase activity, cytotoxic to host cells.

10. Clinical Diseases of P. aeruginosa-I Primary Skin infections: The most common is infection of wounds and burns (blue-green pus). *Fluorescent pigment detected in wounds, burns, or urine by UV fluorescence. Pulmonary infections:P. aeruginosa infections of lower respiratory tract.Tracheobronchitisto severe Necrotizing pneumonia. Colonizationseen in CF patients (diffuse, bilateral bronchopneumonia with necrosis). Meningitis (when introduced by lumbar puncture). Eye infections: corneal ulcer.

11. Clinical Diseases of P. aeruginosa-II Ear infections: External Otitis => Often seen in “Swimmer’s ear” mild in swimmers; malignant (invasive) in diabetic patients. Chronic otitis media Endocarditisseen in intravenous drug abusers. Urinary tract infection Sepsis: most cases originate from infections of lower RT, UT, and skin and soft tissue. *Ecthyma gangrenosum in sepsis: hemorrhagic necrosis of skin, often do not contain pus.

12. Clinical Diseases of P. aeruginosa

13. Outline • Physiology & Structure • Pathogenesis & Immunity • Lab Diagnosis, Treatment, & Prevention

14. Laboratory Diagnosis of P. aeruginosa 1. Specimen: skin lesions, pus, urine, blood, spinal fluid, sputum. 2. Culture: blood agar plate and differential media. Identification of P. aeruginosa is described above. 3. Identification of P. aeruginosa is usually based on colonial morphology, b-hemolysis, oxidase positive, the presence of characteristic pigments and sweet odor, and growth 42 C 4. Several subtyping methods, including phage typing and molecular typing, are available for epidemiologic purposes. Treatment Combined antibiotic therapy is generally required because the bacteria develop resistance rapidly when single drugs are employed.

15. P. aeruginosa Prevention and Control-I 1. Pseudomonas spp. normally inhabit soil, water, and vegetation and can be isolated from the skin, throat, and stool of healthy persons. 2. Spread is from patient to patient via contact with fomites or by ingestion of contaminated food and water. 3. Methods for control of infection are similar to those for other nosocomial pathogens. 4. Special attention should be paid to sinks, water baths, showers, hot tubs, and other wet areas. 5. High risk population: patients with leukemia, burns, cystic fibrosis, and immunosuppression.

16. P. aeruginosa Prevention and Control-II Control: 1. Patients at high risk should not be admitted to a ward where cases of pseudomonas infection are present. 2. Patients infected with P. aeruginosa should be isolated. 3. Sterilize all instruments, apparatus, and dressing; antimicrobial and other therapeutic substances. 4. Monitor clinically relevant isolates of P. aeruginosa by a suitable typing system to identify epidemic strains.

17. Stenotrophomonas maltophilia 1. A common nonfermentative, gram-negative isolate. 2. It infects debilitated or immunocompromised persons, and causes a wide spectrum of diseases, including wound infections, UT infections, pneumonia, sepsis, meningitis, etc. 3. It is resistant to many commonly used antibiotics, and patients receiving long-term antibiotic therapy are particularly at risk for acquiring infections. 4. Infections may be acquired from contaminated disinfectants, respiratory therapy and monitoring equipment, and ice machines.

18. Burkholderia 1.They colonize the moist environmental surfaces and are commonly associated with nosocomial infections. 2. B. cepacia and B. pseudomalei are important pathogens. 3. B. cepacia causes RT infections (particularly in cystic fibrosis patients), UT infections and septicemia. Usually non-fatal except for RT infections in CF patients. 4. B. pseudomalei usually causes opportunistic infections, but may sometimes infect previously healthy persons. Infection by this organism may result in asymptomatic infection, acute suppurative cutaneous infection that may progress to sepsis, and chronic pulmonary infection ranging in severity from mild bronchitis to necrotizing pneumonia.

19. 2005/7/30 台南高雄疑似發生類鼻疽疫情，疾病管制局提醒民眾，皮膚如有傷口，請勿接觸污染的土壤或水源 疾病管制局今天公佈今年自七月11日至29日以來，類鼻疽累計通報16例，其中高雄縣9例、台南市4例、高雄市2例、台南縣1例。其中6例死亡，3例在加護病房，另7例住普通病房。類鼻疽係由類鼻疽伯克氏菌Burkholderia pseudomallei所造成的臨床感染症，屬假單胞菌屬革蘭氏陰性桿菌，此菌在土壤、水池及積水環境中存在，會感染馬、羊、豬等動物以及人類。其流行地域為東南亞地區及澳洲北部的熱帶地域。該局自89年即將此病納入監測。89年通報病例1例、90年15例、91年9例、92年5例、93年13例。本次疫情發生原因，疾病管制局初步調查研判可能係因日前南部豪大雨，將土壤中之病菌沖刷出來，所造成的民眾感染事件，病例多發生在二仁溪流域。該局鄭重呼籲在二仁溪流域附近居民，若有發燒等症狀者，務必迅速就醫。並告訴醫師居住地區，疾病管制局呼籲，醫師對於上述地區發燒病患，應先排除感染此病的可能性，若有懷疑應立即以抗生素治療，並採檢送驗。

20. Legionella

22. Legionella L. pneumophila: Legionaire's disease; Pontiac fever Morphology and identification Fastidious aerobic gram-negative bacillus. This organism is not seen in stains of clinical specimens, but gram-stain can be made for those grown on agar media. Basic fuchsin should be used as the counterstain. Requires L-cysteine for growth; grows slowly on complex media, such as BCYE (buffered charcoal-yeast extract) agar, at pH 6.9, 32 oC, and 90% humidity. L. pneumophila is nonfermentative, catalase-positive, oxidase-positive, and produces gelatinase and b-lactamase.

23. Antigens and cell products Serogroup 1 is the most common isolate from humans. Produces proteases, phosphatase, lipase, DNase, RNase, hemolysin and cytotoxin. Epidemiology Legionellae are ubiquitous in warm moist environments, such as air-conditioning systems and washing facilities. Legionellae can parasitize ameba in water; they are relatively resistant to heat and disinfectants. Legionellosis, sporadic or epidemic, is distributed worldwide. The elderly are at greatest risk. Person-to-person spread or an animal reservoir has not been demonstrated. Contact with the organism and acquisition of immunity after an asymptomatic infection are common.

24. Legionella spp. Associated with human disease

25. Pathology and Immunity L. pneumophila Infects mainly debilitated or immunocompromised humans following inhalation of the bacteria from aerosols generated from contaminated sources, and causes acute purulent pneumonia. L. pneumophila is facultative intracellular parasite, readily enters (via CR3 complement receptor) macrophages and monocytes; inhibits phagolysosomal fusion; and multiplies in the vacuole until the host cells is lysed. It is not effectively killed by PMNs, also. Immunity to disease is primarily cell-mediated, with humoral immunity playing a minor role.

26. Clinical Diseases Risk factors: compromised pulmonary functions (e.g., smoking, chronic bronchitis and emphysema); decreased cellular immunity (e.g., elderly persons, chemotherapy, steroid and other immunosuppressants, and diabetes mellitus). Legionair's disease (legionellosis): typically severe, rapidly progressive illness with high fever, chills, malaise, nonproductive cough, hypoxia, diarrhea, and delirium. Multiorgan involvement is common. Primary manifestation: pneumonia with multilobar consolidation and inflammation and microabscesses in lung tissue. Pontiac fever (influenza-like illness): self-limited; fever and chills, myalgia, malaise, and headache, but no evidence of pneumonia. A hypersensitivity reaction to the organism.

27. Laboratory Diagnosis Microscopic examination: direct fluorescent antibody (DFA) test. Specific, but sensitivity is low. Culture: use BCYE supplemented with antibiotics to suppress rapidly growing contaminating bacteria. Legionellae grow after 3 to 5 days. Identification of Legionella is by the finding of typical morphology and specific growth requirements. Urinary antigen tests: detection of Legionella-specific LPS antigens secreted in the urine of patients by EIA. Serology: Detection of antigen requires serogroup-specific reagents, however, only serogroup-1 antibodies is commercially available. Antibodies can be detected by indirect fluorescent antibody test.

28. Treatment, Prevention, and Control Macrolides or fluoroquinolones are used to treat severe Legionella infections. Pontiac fever is self-limited and requires no specific therapy. Prevention of legionellosis requires identification of the environmental source of the organism and reduction of the microbial burden. Chlorination and heating of water, and cleaning of the air-conditioning systems are usually effective for prevention and control of legionellosis.

29. 最近的疫情 【大紀元2005年9月1日報導】(據明報新聞網報導)西班牙東北部薩拉戈薩市上月爆發退伍軍人症，目前共有21人染病，2人死亡。 西班牙衛生部說，薩拉戈薩市8月1日開始發現退伍軍人症病例，隨後疫情出現蔓延趨勢。目前仍有16人留醫，其中2人病情嚴重。 薩拉戈薩市政府有關部門目前還沒有找到引發退伍軍人症的原因。當地已拆除了64座可能含有退伍軍人症病菌的水塔。 去年，薩拉戈薩市也曾發生退伍軍人症疫情，造成32人染病，7人死亡。 退伍軍人症是一種傳染性比較強的細菌性肺炎，由孳生在空氣加濕器、蓄水系統、空調系統等潮濕環境中的退伍軍人菌引起，主要症狀是高燒、咳嗽和腹瀉，患者死亡率約10%。體弱者和老年人最易受感染。 ×

30. 台灣退伍軍人症的發展史 台灣早期對此病之認識不夠，常被當做一般肺炎誤診，民國七十八年首先自美日引進嗜肺退伍軍人菌之檢驗技術，八十二年曾發生醫院工作人員感染，導致腎衰竭而需長期洗腎；八十四年更有因本症死亡之病例，使本病更受台灣醫界的重視。 台灣地區退伍軍人桿菌環境檢測概況 臺灣根據行政院衛生署檢疫總所對供水系統做調查，結果顯示出，退伍軍人桿菌在不同給水系統中污染程度約呈常態分佈，大約30%屬於未受退伍軍人桿菌污染或輕微污染的(<10cfu/mL)，約60%屬於低或中程度的污染(10～100cfu/mL)，只有10%是受到較高度污染的(100～1,000 cfu/mL)。台北市衛生局於1997年作抽樣調查中發現所調查場所的蓮篷頭大約有11.85%不合乎標準，游泳池約有25%不合乎標準(檢體數過少)；池水約有5.88%不合乎標準。再就醫院而言大約有48%醫院之冷卻水塔不合乎標準；7.6%水龍頭不合乎標準；飲水機不合乎標準大約有18.8%；蓮篷頭大約有13.46%不合乎標準。由中國附設醫院調查冷卻水塔退伍人桿菌水質檢驗結果發現44%電子公司、25%醫院、29%戲院、23%餐館其冷卻水塔超過標準。 (摘自感染控制雜誌第12卷第2期)