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)

Outline

1. Physiology & Structure

2. Pathogenesis & Immunity

3. Lab Diagnosis, Treatment, & Prevention

Structure and Physiology1. 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)

Pseudomonas (pseudes, false; monas, a unit or monas)

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

P. Aeruginosa (refer to green pigment)

Forms round colonies with a fluorescent greenish color, sweet odor, and -hemolysis.

Pyocyanin- nonfluorescent bluish pigment;

pyoverdin- fluorescent greenish pigment;

pyorubin, and pyomelanin

Some strains have a polysaccharide capsule.

Identification of P. aeruginosa is usually based on colonial m

orphology (flat colonies w/ spreading border), -hemolysis, o

xidase positive, the presence of characteristic pigments and

sweet odor, and growth at 42 oC.

Outline

1. Physiology & Structure

2. Pathogenesis & Immunity

3. Lab Diagnosis, Treatment, & Prevention

P. aeruginosaPathogenesis 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.

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.

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.

Virulence Factors of P. aeruginosa-II

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. Tracheobronchitis to severe Necrotizing pneumonia. Colonization seen in CF patients (diffuse, bilateral bronchopneumonia with necrosis).

Meningitis (when introduced by lumbar puncture).Eye infections: corneal ulcer.

Clinical Diseases of P. aeruginosa-I

Ear infections:External Otitis => Often seen in “Swimmer’s ear”

mild in swimmers; malignant (invasive) in diabetic patients. Chronic otitis media

Endocarditis seen 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.

Clinical Diseases of P. aeruginosa-II

Clinical Diseases of P. aeruginosa

Outline

1. Physiology & Structure

2. Pathogenesis & Immunity

3. Lab Diagnosis, Treatment, & Prevention

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.

Laboratory Diagnosis of P. aeruginosa

Prevention and Control-I1. 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.

P. aeruginosa

Prevention and Control-IIControl: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. Sterilizeall 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.

P. aeruginosa

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 infectio

ns, UT infections, pneumonia, sepsis, meningitis, etc.

3. It is resistant to many commonly used antibiotics, and patie

nts receiving long-term antibiotic therapy are particularly at ris

k for acquiring infections.

4. Infections may be acquired from contaminated disinfectant

s, respiratory therapy and monitoring equipment, and ice ma

chines.

Burkholderia1.They colonize the moist environmental surfaces and are com

monly 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 excep

t for RT infections in CF patients.

4. B. pseudomalei usually causes opportunistic infections, but m

ay sometimes infect previously healthy persons. Infection by thi

s organism may result in asymptomatic infection, acute suppura

tive cutaneous infection that may progress to sepsis, and chroni

c pulmonary infection ranging in severity from mild bronchitis to

necrotizing pneumonia.

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。

Legionella

LegionellaL. pneumophila: Legionaire's disease; Pontiac fever

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 -lactamase.

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.

Serogroup 1 is the most common isolate from humans.

Produces proteases, phosphatase, lipase, DNase, RNase, hemolysin and cytotoxin.

Antigens and cell products

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.

Epidemiology

Legionella spp. Associated with human disease

L. pneumophila Infects mainly debilitated or immunocompromise

d humans following inhalation of the bacteria from aerosols gene

rated from contaminated sources, and causes acute purulent pne

umonia.

L. pneumophila is facultative intracellular parasite, readily enters

(via CR3 complement receptor) macrophages and monocytes; in

hibits phagolysosomal fusion; and multiplies in the vacuole until t

he host cells is lysed. It is not effectively killed by PMNs, also.

Immunity to disease is primarily cell-mediated, with humoral imm

unity playing a minor role.

Pathology and Immunity

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.

Clinical Diseases

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.

Laboratory Diagnosis

Macrolides or fluoroquinolones are used to treat severe

Legionella infections.

Pontiac fever is self-limited and requires no specific ther

apy.

Prevention of legionellosis requires identification of the

environmental source of the organism and reduction of t

he microbial burden. Chlorination and heating of water,

and cleaning of the air-conditioning systems are usually

effective for prevention and control of legionellosis.

Treatment, Prevention, and Control

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。

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 )