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Bacteria

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Pasteurellaceae, Legionellaceae
Sung‐Pin Tseng (曾嵩斌), Ph. D.
Department of Medical Laboratory Science and Biotechnology, KMU ext: 2353 E‐mail: tsengsp@kmu.edu.tw

Outline (課程要點與學習目標)
Characteristic of Pasteurellaceae, Legionellaceae
Infections produced by pathogenic species
Tests used to identify these species
Reference:
Chapter 34, 37 Medical Microbiology, 6th Edition

Department of Medical Laboratory
Science and Biotechnology, KMU

Department of Medical Laboratory
Science and Biotechnology, KMU

Pasteurellaceae (巴斯德桿菌科)
 Classification – includes four medically

important genera

Haemophilus Influenzae
 Misnamed – originally thought to cause the “flu”


Haemophilus
 Pasteurella
 Aggregatibacter
 Actinobacillus (rare clinical isolate)




Now know that flu is caused by viruses
In some cases of flu, H. influenzae is secondary infection

 Small, G(‐), aerobic or facultative anaerobic

rods, requiring enriched media for isolation
Department of Medical Laboratory
Science and Biotechnology, KMU

small, cocobacilli form in sputum from patient with pneumonia thin, pleomorphic form (多形性) from patient with meningitis (80%)
Department of Medical Laboratory
Science and Biotechnology, KMU

Haemophilus Species
 Haemophilus = “blood loving”
 Haemophilus spp require hemoglobin for growth:


X‐factor ( hemin)





Satellite phenomenon
H. Influenzae (small colonies surrounding
S. aureus colonies)

Heat‐stable substance
Present in RBC and released with degradation of hemoglobin

V‐factor (NAD: nicotinamide adenine dinucleotide)


Heat‐ labile, coenzyme I, found in blood or secreted by certain organisms  On blood agar H. influenzae exhibits satellitism around colonies of Staphylococcus aureus (a source of V factor).
Department of Medical Laboratory
Science and Biotechnology, KMU

Virulence factors of
Haemophilus influenza (I)
Capsule: six serotypes (a to f); antiphagocytic
 Noncapsular Haemophilus spp commonly colonized humans  H. influenza serotype b cause >95% invasive infection
H. influenza serotype b have an antiphagocytic polysaccharide capsule which contains ribitol, ribose, and phosphate
(polyribitol phosphate; PRP) http://www.ppdictionary.com/bacteria/gnbac/influenzae.htm Department of Medical Laboratory
Science and Biotechnology, KMU

S. aureus excretes
V factor (NAD) into medium

Department of Medical Laboratory
Science and Biotechnology, KMU

Virulence factors of
Haemophilus influenza (II)
Fimbriae increase the adherence of bacteria to human mucosal cells in vitro lipopolysaccaride lipid A induced meningeal inflammation in animal model & human
IgA1 proteases facilitate colonization on mucosal surfaces Department of Medical Laboratory
Science and Biotechnology, KMU

Infections by Haemophilus influenza (I)

Infections by Haemophilus influenza (II)

Disease caused by H. influenzae usually begins in the upper respiratory tract as nasopharyngitis http://medicsindex.ning.com/profiles/blogs/respiratory‐tract‐infection Department of Medical Laboratory
Science and Biotechnology, KMU

Clinical diseases of
H. influenza

Conjunctivitis (結膜炎)

http://medicsindex.ning.com/profil es/blogs/respiratory‐tract‐infection Department of Medical Laboratory
Science and Biotechnology, KMU

DNA Replication

Sinusitis (鼻竇炎)

epiglottitis (會厭炎) http://www.medicinenet.com/cellulitis/article.htm Department of Medical Laboratory
Science and Biotechnology, KMU

Department of Medical Laboratory
Science and Biotechnology, KMU

Hib incidence rate per 100,000 children under five years

Bacterial meningitis caused by Haemophilus influenzae, Neisseria meningitidis and
Streptococcus pneumoniae prior to 1985
Vaccine not available

http://www.docstoc.com/docs/44432145/Hib‐incidence‐rate‐per‐
100000‐children‐under‐five‐years

Department of Medical Laboratory
Science and Biotechnology, KMU

The decline of Hib meningitis associated with the introduction of new vaccines, USA

http://textbookofbacteriology.net/themicrobialworld/haemophilus.html

Department of Medical Laboratory
Science and Biotechnology, KMU

The Gambia Hib Vaccine Program
Resulted in Virtual Disease Elimination

Attaching Hib polysaccharide to a protein carrier greatly increased the ability of the immune system of young children to recognize the polysaccharide and develop immunity

Department of Medical Laboratory
Science and Biotechnology, KMU

http://www.prb.org/Articles/2006/HibVaccineCouldReduceMaj orChildhoodDiseases.aspx Department of Medical Laboratory
Science and Biotechnology, KMU

Global Status of Countries Using Hib Vaccine in Their National Immunization System, 2004

Hib Vaccine not recommended in Taiwan
2000 -2004 年0-10 歲兒童發生
Hib確定病例的機率為每十萬人
中5.225~12.805 個病例

http://www.prb.org/Articles/2006/HibVaccineCouldReduceMaj orChildhoodDiseases.aspx Department of Medical Laboratory
Science and Biotechnology, KMU

Increasing Incidence of Invasive Haemophilus influenzae Disease in Adults, Utah, USA

台灣5 歲以下兒童b 型嗜血桿菌(Hib)
疫苗接種之成本效益分析;佘紫瀅; 96

Department of Medical Laboratory
Science and Biotechnology, KMU

Increasing Incidence of Invasive Haemophilus influenzae Disease in Adults, Utah, USA

Black line, Intermountain Healthcare red line, Utah Department of Health

http://wwwnc.cdc.gov/eid/article/17/9/10‐1991‐f1.htm

Department of Medical Laboratory
Science and Biotechnology, KMU

http://wwwnc.cdc.gov/eid/article/17/9/10‐1991‐f3.htm

Department of Medical Laboratory
Science and Biotechnology, KMU

Infection of Haemophilus ducreyi

Incidence of Haemophilus ducreyi

Causative agent of chancroid (軟性下疳) or soft chancre (STD)
A sexually transmitted disease;
Most commonly diagnosed in men

http://www.health‐reply.com/chancroid‐ granuloma‐inguinale‐lymphogranuloma/ http://doctorsgates.blogspot.com/2011/02/clinical‐symptoms‐of‐haemophilus.html

Department of Medical Laboratory
Science and Biotechnology, KMU

Comparison of chancroid & syphilis incidence in united states army, 1908‐30

http://www.scielosp.org/pdf/bwho/v79n9/v79n9a06.pdf

Department of Medical Laboratory
Science and Biotechnology, KMU

Department of Medical Laboratory
Science and Biotechnology, KMU

Chancroid incidence rates in USA, 1941‐98

http://www.scielosp.org/pdf/bwho/v79n9/v79n9a06.pdf

Department of Medical Laboratory
Science and Biotechnology, KMU

Major causes of genital ulcers

Infection of Haemophilus aegyptius

Conjunctivitis (結膜炎)

http://www.ncbi.nlm.nih.gov/books/NBK8136/

Department of Medical Laboratory
Science and Biotechnology, KMU

Haemophilus species associated with human diseases
Species
H. influenzae

H. aegyptius
H. ducreyi
H. parainfluenzae

Primary diseases
Pneumonia; sinusitis; otitis; meningitis; epiglottitis; cellulites; bacteremia
Conjunctivitis
Chancroid

Bacteremia; endocarditis; opportunistic infections
H.haemolyticus
opportunistic infections
H. parahaemolyticus opportunistic infections

http://www.eflnet.com/vocab/dictionary/eyes_def?myword=pinkeye

Department of Medical Laboratory
Science and Biotechnology, KMU

Haemophilus influenzae

Frequency
Common

Uncommon
Uncommon
Rare chocolate agar common used

Rare
Rare

Department of Medical Laboratory
Science and Biotechnology, KMU

http://www.microbiologyinpictures.com/haemophilus%20influenzae.html

Department of Medical Laboratory
Science and Biotechnology, KMU

Haemophilus Species: Identification

Porphyrin test

Catalase +; Oxidase +
X and V factors

+
+
+
+


Horse/Rabbit
BAP Hemolysis

+ v +
+/‐

Porphyrin test ‐

+
+






+

Haemophilus spp.

X

V

H. influenzae
H. haemolyticus
H. parainfluenzae
H. parahaemolyticus
H. ducreyi
Aggregatibacter.
aphrophilus

+
+


+
+/‐

Department of Medical Laboratory
Science and Biotechnology, KMU

Haemophilus Species: Identification

Under ultraviolet light, the organism on the bottom is exhibiting a positive porphyrin reaction. The organism on the top is porphyrin negative Department of Medical Laboratory
Science and Biotechnology, KMU

H. parahaemolyticus: Identification
 Although BAP contains both X and V factors, only X is directly available
 V is not available on BAP because sheep blood contains NADase that hydrolyzes V
 Quad plates contain X and V factors & horse blood agar
X

This organism would be identified as H. influenzae because it is using both X and
V factors.

V

This organism would be identified as H. parainfluenzae because it is using V factor only. Horse Blood agar

http://www.microbiologyinpictures.com/bacteria%20photos/haemophilus%20i nfluenzae%20photos/HAIN22.html Department of Medical Laboratory
Science and Biotechnology, KMU

http://faculty.matcmadison.edu/mljensen/111CourseDocs/111Review
/Unit2Reviews/haemophilus_answers.htm

X V

Laboratory Diagnosis
Microscopy: gram‐negative rods, pleomorphic filaments (80% CSF specimens)
Antigen detection: PRP capsular antigen can be detected in CSF & urine (particle agglutination test)
Serotype b meningitis can be diagnosed by detecting capsule Ag
Culture: Chocolate agar (V & X factors);
Levinthal's agar

polyribitol phosphate (PRP)

Satellite phenomenon (with S. arueus) from blood agar

Satellite phenomenon
H. Influenzae (small colonies surrounding
S. aureus colonies)

S. aureus excretes
V factor (NAD) into medium

http://www.ppdictionary.com/bacte ria/gnbac/influenzae.htm Department of Medical Laboratory
Science and Biotechnology, KMU

Department of Medical Laboratory
Science and Biotechnology, KMU

Treatment

Prevention & control

Historically ampicillin was the drug of choice.
However, resistance has developed due to production of beta‐lactamase or altered penicillin binding proteins and cell wall permeability Serious infections are treated with broad‐ spectrum cephalosporins including cefotaxime or ceftriaxone
H. ducreyi are susceptible to erythromycin

Active immunization with purified capsular PRP
Three doses of vaccine against H. influenzae type b disease before the age of 6 months, followed by booster doses.
Antibiotic chemoprophylaxis (抗生素預防性投藥) is used to eliminate the carriage of H. influenzae type b in children at high risk for disease
Rifampin prophylaxis has been used in these settings Department of Medical Laboratory
Science and Biotechnology, KMU

Department of Medical Laboratory
Science and Biotechnology, KMU

Movie of Haemophilus

HACEK GROUP
 Haemophilus species
 Aggregatibacter actinomyetemcomitas
 Aggregatibacter aphrophilus
 Cardiobacterium hominis
 Eikenella corrodens
Old name: Actinobacillus
 Kingella species actinomyetemcomitas http://www.youtube.com/watch?v=ogqrgd3i_mQ&feature=related

Department of Medical Laboratory
Science and Biotechnology, KMU

General Characteristics
Gram‐negative bacilli
 Require an increased CO2 (5%‐10%) environment
 Slow/poor growers
 Usual flora of the oral pharyngeal cavity
 Opportunists in immunocompromised hosts


Department of Medical Laboratory
Science and Biotechnology, KMU

Department of Medical Laboratory
Science and Biotechnology, KMU

Clinical significance
 Infective endocarditis
 Most common cause of gram negative endocarditis in non drug users
 Also associated with
 Periodontal infections, Bacteremia, Abcesses,
Peritonitis, Otitis media, Conjunctivitis, Septic arthritis, Osteomyelitis, UTI, Brain abscess
 Infections frequently associated with dental procedure (why?)
Department of Medical Laboratory
Science and Biotechnology, KMU

Clinical characteristics of Aggregatibacter actinomycetemcomitans from NTUH in Taiwan

Pasteurellaceae (巴斯德桿菌科)
 Classification – includes four medically

infective endocarditis important genera
Haemophilus
 Pasteurella
 Aggregatibacter
 Actinobacillus (rare clinical isolate)


 Small, G(‐), aerobic or facultative anaerobic

rods, requiring enriched media for isolation
J Microbiol Immunol Infect 2010;43(6):491–497

Department of Medical Laboratory
Science and Biotechnology, KMU

Pasteurella
small Gram‐negative bacillus
 fermentative, but anaerogenic, non‐motile, and oxidase +
 P. multocida is the most commonly isolated species  The organisms are usually associated with animals other than man

http://www.infoescola.com/doencas/pasteurelose/

Department of Medical Laboratory
Science and Biotechnology, KMU

Department of Medical Laboratory
Science and Biotechnology, KMU

Infection of Pasteurella multocida
Pasteurella multocida is a bacterium that's commonly found in the mouths of dogs and cats. It's a common cause of cat and dog bite infections in people, but can also be spread through close contact with pets (without bites).

http://www.wormsandgermsblog.com/tags/pasteurella‐multocida/

Department of Medical Laboratory
Science and Biotechnology, KMU

Pasteurella spp associated with human disease

Treatment of Pasteurella
Antimicrobial sensitivity is exquisitely sensitive to penicillin
Tetracycline and chloramphenicol may also be used

Department of Medical Laboratory
Science and Biotechnology, KMU

Ch 37

General Overview of Legionellaceae
Aquatic, aerobic, thin G(‐) bacilli; Facultative

intracellular parasites
Nutritionally fastidious; require L‐cysteine; additional iron enhance growth
Slow grower, need 3‐10 days to form colonies
At least 46 species of Legionella have been identified.
More than one‐half species have implicated as human pathogen. 80~90% are caused by Legionella pneumophila (嗜肺性退伍軍人菌), and serogroups 1,4, 6 are most commonly.
Department of Medical Laboratory
Science and Biotechnology, KMU

Department of Medical Laboratory
Science and Biotechnology, KMU

Murine bone‐marrow‐derived macrophages were infected with the virulent L. pneumophila
The macrophages were fixed and stained for L. pneumophila
(red) and LAMP‐1 (lysosomal membrane protein LAMP‐
1;green)
Large arrows identify two yeast phagosomes that each show intense staining for the
LAMP‐1 protein on the surrounding membrane http://www.nature.com/ncb/journal/v1/n7/pdf/ncb1199_451.pdf Department of Medical Laboratory
Science and Biotechnology, KMU

Legionella species associated with human disease
L. micdadei is the most common after L. pneumophila. L. bozemanii is more virulent or resistant than
L. pneumophila

History of Legionella (退伍軍人菌)
Legionnaires’ disease was first recognized in
1976, when an outbreak of pneumonia took place at a hotel in Philadelphia during the
American Legion Convention (美國退伍軍人協會) .

What kind infectious way of Legionella ?
Department of Medical Laboratory
Science and Biotechnology, KMU

Department of Medical Laboratory
Science and Biotechnology, KMU

Risk factors for Legionella infection

http://osha.europa.eu/en/publications/literature_reviews/le gionella‐policy‐overview.pdf Department of Medical Laboratory
Science and Biotechnology, KMU

Department of Medical Laboratory
Science and Biotechnology, KMU

Epidemiology of Legionella

The environmental life of L. pneumophila within protozoa

Capable of sporadic, epidemic & nosocomial infections Commonly found in natural bodies of water, cooling towers, condensers, & water systems
(including hospital systems)
Patients at high risk for symptomatic disease include patients with compromised pulmonary function & patients with decreased cellular immunity (particularly transplant patients)
Department of Medical Laboratory
Science and Biotechnology, KMU

Rates of legionellosis in the world

M. Molmeret et al. Microbes and Infection 6 (2004) 129–139

Comparison of disease caused by Legionella
Severe pneumonia

Department of Medical Laboratory
Science and Biotechnology, KMU http://www.gideononline.com/tag/legionellosis/ Department of Medical Laboratory
Science and Biotechnology, KMU

Influenza‐ like illness

Severe pneumonia

Influenza‐ like illness

Department of Medical Laboratory
Science and Biotechnology, KMU

Laboratory Diagnosis I
Microscopy: stain poorly with gram stain; direct fluorescent antibody (DFA)
Nucleic acid amplification assays Culture: buffered charcoal‐yeast extract (BCYE agar) 35℃, 3‐5 days

Urinary antigen test: Detection of Legionella serogroup 1‐specific lipopolysaccaride antigens in urine Antibody detection: antibody titer 1:128 or greater Department of Medical Laboratory http://pathmicro.med.sc.edu/ghaffar/bord‐hemo.htm Laboratory Diagnosis II

Science and Biotechnology, KMU

Treatment, Prevention, & Control
Clinical experience: Macrolides or fluroquinolones β‐lactam are ineffective because most isolates produce β‐lactamses
Specific therapy for Pontiac fever is generally unnecessary, because it is a self‐limited hypersensitivity disease
Prevention of legionellosis requires identification of the environmental source of the organism & reduction of microbial burden
Department of Medical Laboratory
Science and Biotechnology, KMU

Department of Medical Laboratory
Science and Biotechnology, KMU

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