Lecture 9: Small Gram Negative Bacilli, Section C

I. HEMOPHILUSMORPHOLOGYPleomorhic, coccobacilli 0.2 – 0.5 – 0.8 um with faint refractile capsules Demonstrable by quellung Gram negative but may appear gram variable May exhibit bipolar staining

Haemophilus spp.

PHYSIOLOGY CULTURE MEDIA

1. Chocolate agar (heated blood) to 80°C. Factor X and V released2. Levinthal enriched agar - differentiating - capsulated from non-capsulated3. Fildes agar4. Blood agar used if X-inoculated with Staphylococcus

Incubation at 10% CO2 recommended Growth apparent after 18-24 hours

- colonies 0.5 X 1.5 mm, encapsulated varieties produce glistening, mucoid colonies that are iridescent on Levinthal agar- convert spontaneously to rough colonies- tendency to autolyze

HAEMOPHILUS INFLUENZA ON CHOCOLATE AGAR

HAEMOPHILUS INFLUENZA ON CHOCOLATE AGAR (close-up view)

IDENTIFICATION OF HAEMOPHILUS INFLUENZA BY X AND V STRIPS

Characteristics and growth requirements of some Hemophilus species (X = heme; V =

nicotinamide-adenine dinucleotide)

SpeciesRequire

s HemolysisX V

H. influenzae (H. aegyptius)

+ + -

H. parainfluenzae - + -H. ducreyi + - -

H. haemolyticus + + +H. parahaemolyticus - + +H. aphrophilus - - -ANTIGENIC STRUCTURE1. Capsular polysaccharide – 6 antigenic types (a-f)2. Lipopolysaccharide3. Outer member proteinMAJOR VIRULENCE FACTORS1.Polysaccharide capsule (PRP)

polyribitol phosphate – induce natural immunity after infection or vaccination

2. Lipopolysaccharide lipid A – induces meningeal irritation3. IgA, specific proteases – facilitate colonization of

mucosal surfaces4. Outer-membrane proteins and pili – poorly defined roles

EPIDEMIOLOGY OF HAEMOPHILUS INFECTION Disease/ Bacterial Factors

- H. influenzae type b causes meningitis, epiglottitis, cellulites, arthritis, conjunctivitis

- H. influenzae type b is primarily a pediatric pathogen (children younger than 5 y/o), although the incidence is rapidly decreasing with immunization

- H. ducreyi is an important cause of genital ulcer

- Nonencapsulated H. infuenzae type b causes otitis media, sinusitis, bronchitis

- The type b polysaccharide capsule is ANTIPHAGOCYTIC

- Increased exposure to nonencapsulated Hemophilus strains is not associated with increased infections because these organisms are ubiquitous and have a low virulence

Transmission- Endogenous spread from upper respiratory

tract through the blood to meninges, epiglottis, skin, joints; or direct extension to eye, inner ear, sinuses, or lower respiratory tract

- H. ducreyi: sexually transmitted

Who are at risk?1.children < 5 years old without protective

antibodies against type-b capsular polysaccharide

2. patients with depleted complement of after splenectomy

3. elderly at greatest risk for pulmonary disease, particularly those with obstructive pulmonary disease or conditions predisposing to aspiration

Geography/ seasonH. influenzae infections occur worldwideH. ducreyi genital ulcers in Africa and Asia, less commonly in Europe and North AmericaNo seasonal incidence

LABORATORY DIAGNOSIS1. Gram staining2. Culture – blood culture should be performed

on any patient with meningitis or other invasive disease

3. Antigen detection- CIE – counterimmunoelectrophoresis- latex particle agglutination- ELISA

Recommended for patients with negative culture

TREATMENTA. ANTIMICROBIALS

1. Meningitis – Chloramphenicol/+ Ampicillin2. Otitis media – Amoxycillin, TMP-SMX,

Erythromycin, Cefaclor3. Sinusitis – Ampicillin

B. PASSIVE IMMUNOTHERAPY adjunct to antibiotics – in treatment of severe

infection

PREVENTION1. Active immunization

1985 – Purified capsular polysacc vaccine protective for 2 year old

* Conjugated vaccines – with diphtheria and Neisseria meningitides

2. Passive ImmunizationChildren with congenital or acquired deficiencies

3. Chemoprophylaxis – Rifampicin – Controversial

OTHER HEMOPHILUS SPECIES1. Haemophilus aegyptius (Koch-weeks)

- associated with purulent conjunctivities especially in children

2. H. para-influenza – part of the normal flora, causes of opportunistic infections. Rare cause of meningitis

3. H. ducreyi – STD, worldwide in distribution, responsible for 10% of venereal diseases in temperate countries, more common among non-whites and in poor socio-economic and hygienic condition indurated, painful, single or multiple lesions, developing in the genitals and perianal areas, accompanied by suppurative inguinal buboes.

II. BORDETELLA3 Species Disease

B. pertussis (man is only natural host

Pertussis or whooping cough

B. parapertusis (man is only natural host)

Milder form of whooping cough

B. bronchisepticaBronchopulmonary disease in animals and humans

PHYSIOLOGY AND STRUCTURE Size: 0.2 – 0.5 X um

Strictly aerobic, gram negative coccobacilli non-motile, highly communicable, multiply among cilia of epithelial cells

Culture medium modified Bordet-Gengou medium (potato-

glycerol-blood agar) Regan- Lowe-medium-charcoal, oxoid

medium with horse blood Stainer – scholte agar – selective medium,

containing cyclodextrin and cephalexin Colonies are pinpoint in size, smooth convex,

glistening, almost transparent and pearl like, produce zone of hemolysis that varies with cultural conditions.

Variation manifested with change from phase 1 – IV variant1. Serial passage on culture media

2. High level of magnesium in media

B. bronchiseptica on 5% sheep blood agar

Differential Characteristics of Bordetella SpeciesBordatella species

Characteristics

B. pertussis

B. parapertussis

B. bronchiseptica

Oxidase + - +Urease - + +Motility - - +Browning on:Mueller-Hinton Agar - + -

Growth on:Sheep AgarMacConkey Agar

-

-

+

+

+

+

Virulence Factors Associated with Bordetella pertussis

Virulence Factors Biological Effects

Pertussis toxins

ADP-ribosylation of guanine nucleotide-binding proteins, lymphocytosis, hypoglycemia, mediates attachment of respiratory epithelium

Adenylate cyclase toxin

Impairment of leukocyte chemotaxis and killing, local edema

Tracheal cytotoxinCiliastasis and then extrusion of ciliated epithelial cells

Dermonecrotic toxinVascular smooth muscle contraction and ischemic necrosis

Filamentous hemaglutinin

Mediates attachment to ciliated epithelial cells, agglutinates erythrocytes

Lipopolysaccharide Exotoxin activity

CLINICAL PRESENTATION OF B. PERTUSSIS DISEASE

Incubation

Catarrhal

Paroxysmal Convalescent

Duration 7-10 days 1-2 weeks 2-4 weeks 3-4 weeks (or

longer)

Symptoms None

Rhinorrhea, malaise, fever, sneezing, anorexia

Repetitive cough with whoops, vomiting, leokocytosis

Diminished paroxysmal cough, development of secondary complications (pneumonia, seizures, encephalopathy)

Bacterial Culture

LABORATORY DIAGNOSIS

Specimen – optimum is nasopharyngeal aspirate

(organism very sensitive to drying, fatty acids on cotton swabs are toxic)

- Direct inoculation on suitable medium recommended.

MICROSCOPY DFA – direct flourescent antibody

CULTURE (as long as 7 days) tiny colonies are observed only after 3 days

III. Francisella tularensis

- causative agent of tularemia (glandular fever, rabbit fever, tick fever, deerfly fever) both in rodents and humans

A. Physiology and Structure - size: 0.2 x 0.2-0.7 um

- sometime bipolar on polychrome, faintly staining- gram negative coccobacilli- non-motile, non-piliated, with thin lipid capsule- pleomorphic obligate aerobe- weakly catalase positive- biochemical characterization of little value in identification

Culture Characteristics- growth at 24°C- 39°C- slow growing- requires cystine or cysteine- colonies noted after 2-4 days incubation on glucose- cysteine clood agar (CGB+) or peptone cysteine agar (RCA) - greenish disc surrounds the colony but no true hemolysis occurs

B. Epidemiology of Tularemia Disease/ Bacterial Factors

- Ulceroglandular, glandular, typhoidal, oculoglandular and oropharyngeal tularemia

- F. tularensis is a facultative intracellular pathogen and can survive for prolonged periods in macrophages of the reticuloendothelial system

- Antiphagocytic capsule is present in pathogenic strains- Most common reservoirs in the United States are

rabbits, ticks, and muskrats Transmission

- Bite from infected tick or contact with infected animals

- Infection also acquired after ingestion of contaminated meat or water, or inhalation of an infectious aerosol

Who are at risk?- hunters - persons exposed to ticks- laboratory personnel

Geography/ Season - Worldwide; in the United States most infections are

in Arkansas, Missouri, and Oklahoma- Disease most frequent during summer and winter

C. Manifestations of TularemiaType of disease

Infections (%)

Characteristics

Ulceroglandular 75-85

Ulcers at the site of exposure and adenopathy of the draining lymph nodes

Glandular 5-10Adenopathy but no ulcers

Typhoidal 5-15

Systemic signs but no adenopathy or ulcers

Oculoglandular 1-2 Eye involvement

Oropharyngeal <1Orapharyngeal involvement

D. Laboratory Diagnosis Specimen collection – hazardous for both physician

and laboratory worker- small organism can penetrate thru the skin and

mucous membrane Microscopy – direct staining of clinical specimen with

flourescin labeled antibodies Cultures

- prolonged incubation specimen – sputum and aspirates from lymph nodes or draining sinuses

- blood culture are usually negativeE. Modes of Control1) Streptomycin (drug of choice), gentamicin2) Avoid reservoirs and vectors infection(e.g., rabbits, ticks)3) Live, attenuated vaccines reduce severity of disease4) Lab personnel: use of gloves and biohazard hood

when handling specimens

IV. Brucella- Bang’s disease- Malta fever- Undulant fever- Mediterranean remittent fever- Rock fever of Gibraltar- Fever of Crete- Country fever of Constantinople- 6 species known, 4 are associated with human

disease – abortus, melitensis, suis, and canis

A. Physiology and Structure Size: 0.5 x 0.6-1.5 um

- non-motile, non-encapsulated- gram negative coccobacilli- grow slowly on culture- aerobic- some strains requiring CO2

- does not ferment lactose- catalase and oxidase (+)- reduce nitrate - with variable urease activity

Gram stain of Brucella bacill

Higher Magnification

Colonies of Brucella sp. on 5% sheep blood agar

Colonies of Brucella spp. on chocolate agar. Growth of Brucella spp. on chocolate agar after 4 days of incubation. Colonies require prolonged incubation to attain sufficient growth for further biochemical testing.

Epidemiology of Brucellosis Disease/ Bacterial factors

- Brucella is a facultative intracellular pathogen and can survive for prolonged periods in macrophages of reticuloendothelial system- Reservoirs are animals: cattle, goats, sheep, swine, dogs, foxes, and coyotes

Transmission - ingestion of contaminated milk or cheese- contact with infected animals

Who are at risk?- persons in direct contact with infected animals:

veterinarians, meat handlers, farmers- persons eating unpasteurized milk or cheese- laboratory personnel

Geography/ Season- worldwide; in the United States, most common in

California and Texas; less disease in the United States attributable to control of disease in animals

- no seasonal incidence

Characteristic of Human Brucellosis Growth on dyes

SpeciesAnimal Reserv

oir

CO2 requir

ed

Basic fuchs

in

Thionin

Clinical disease

B. melitensis

Goats, sheep – + +

Severe, acute disease with complications common

B. abortus Cattle + + –

Mild disease with suppurative complications uncommon

Growth on dyes

SpeciesAnimal Reserv

oir

CO2

required

Basic fuchs

in

Thionin

Clinical disease

B. suis Swine – + +

Suppurative, destructive disease with chronic manifestations

B. conis Dogs – – +

Mild disease with suppurative complications uncommon

Antibody Responses in untreated Brucellosis

C. Laboratory Diagnosis Microscopy – direct fluorescent antibody techniques Culture – slow growing, fastidious MacConkey agar

- 3 or more days identification by biochemical reactions and growth in the presence of basic dyes, fuchsin and thionin

Serology – fourfold increase in antibody titer, IgM followed by IgG and IgA

D. Modes of Control1) Tetracycline combined with streptomycin or with

gentamicin2) Long-term therapy with high doses of

trimethoprimsulfamethoxazole3) Control of disease in animal reservoir4) Avoidance of unpasteurized dairy products5) Protective clothing when working with animals or

specimens

V. Yersinia- consists of 7 species, 3 are best known human

pathogens, the rest can occasionally cause opportunistic infections

- Y. pestis- Y. pseudotuberculosis

- Y. enterocolita

A. Morphology and Physiology- gram negative, non-motile, coccobacillus- marked bipolar staining on tissue impression safety pin appearance- faculatative anaerobes, does not ferment- lactase oxidase (-)- catalase (+)- can grow in ordinary media, small mucoid colonies (on D2 of incubation)- no hemolysis on blood agar

Yersinia pestis in blood (Wright-Giemsa stain)

High Magnification

Strain identification- thru ability to

reduce nitrates- Ferment glycerol

– (orientalis, medievalis and antigua) having differences in geographic distribution

B. Epidemiology of Yersinia infection Disease/ Bacterial factorsY. pestis – plagueY. enterocolitica – enterocolitis, transfusion-related septicemiaY. psuedotuberculosis – enterocolitisY. pestis – present in animal reservoir, fleasOther Yesinia – present in domestic animals (GI tract)

and contaminated food products Numerous virulence factors

Transmission- Y. pestis – spread from mammalian reservoir (rats,

prairie dogs, dogs, mice, rabbits) via fleas or contact with contaminated animal tissues

- Other Yersinia: ingestion of contaminated food products, infusion of contaminated blood products

Who is at risk?- Y. pestis: communities with endemic plague and

exposure to infected animals- Y. enterocolitica: individuals eating contaminated

food, recipients of contaminated blood products

Geography/ Season- Y. pestis : primarily in Asia and Africa- Y. pestis: disease is cyclical, as reservoir

population increases/ decreases- Other Yersinia: infections worldwide but

primarily in cold climates

C. Determinants of Pathigenicity1. Ca2+ dependency2. V and W antigen3. outer membrane proteins4. envelope antigen5. pesticin, coagulase and plasminogen activation6. pigment binding and iron regulated surface proteins

D. Clinical Syndromes1. Bubonic Plague

- 7 days incubation, after bite from infected flea- high fever- painful bubo (inflammatory swelling of lymph node)

groin or axilla- 75% mortality in absence of treatment

2. Pneumonic plague- 2-3 days incubation period- fever, malaise, pulmonary signs within one day- fatality rate – 90% in untreated cases

E. Treatment

Chemotherapy – streptomycin, alternative drugs as tetracyclines

and chloramphenicol- Kanamycin appears as effective as streptomycin

F. Prevention and Control1. Vaccination

– 3 doses, in series recommended to concerned personnel

- laboratory and fields with enzootic plague efficacy of vaccine is also uncertain hence prophylactic antibiotics is recommended for people with definite exposure

2. Quarantine of persons or ships and aircraft carrying persons

3. Flea control before rat control4. Proper garbage disposal and good personal hygiene

VI. Yersinia enterolica and Y. pseudotuberculosis

A. Morphology- motile, with paripolar and

peritrichous- produced at 22°C and not at 37°C- usual media for enteric bacteria- identification based on biochemical differences – the

results are markedly affected by temperature- common cause of enterocolitis in cold countries –

Scandinavia, Europe, and North America- outbreaks associated with contaminated meat or

milk

B. Pathogenic determinants1. Inv (invasion) gene2. Ail (attachment-invasion locus)3. V and W Ag4. Gene for Ca2+ concentration and temperature5. Enterotoxin

– produced by Y. enterocolitica- role still uncertain, but physico-chemical and

antigenic properties is similar to that of heat stable of E. coli

C. Clinical Syndrome1. Diarrhea, fever and abdominal pain lasting for as

long as 1-2 weeks2. Chronic form can develop and persist for months to

one year3. Involves the ileum, with enlargement of mesenteric

lymph nodes. Can mimic acute appendicitis4. Other manifestations may include septicemia,

arthritis, intraabdominal abscess, hepatitis and osteomyelitis

5. 1087 – first reported cause of BT and related Bacteremia and endotoxic shock

D. Diagnosis1. Culture of organisms from uncontaminated samples2. Widal type agglutination test

E. Treatment1. Aminoglycosides2. Trimetoprim and Sulfamethoxazole3. Supportive – fluid and electrolytes

VII. Pastuerella multocida- primarily parasite of domestic animals and birds –

but capable of a variety of human diseases

A. Morphology and Physiology

Size: 0.3-1 um x 1-2 um- gram negative- show bipolar staining - pleomorphic in primary cultures- produce capsule

Facultatively anaerobic- catalase and oxidase positive- metabolism is fermentative

Cultural Characteristics- non-hemolytic on blood agar- does not grow on bile containing media- show round grayish colonies 1-3 minutes within 24

hours Antigenic Structure

- 4 different serotypes (A, B, D, E)- A and D are most associated with human infections

B. Epidemiology- Present as normal flora in domestic animals (dogs and cats)- Transmitted by direct contact, usually animal bite- Those causing respiratory tract infections in man are

caused by Type A

C. Clinical Manifestations1. Infection via bites or scratches2. Superinfection of chronically diseased lung3. Other foci of infection secondary to septicemia

D. Laboratory Diagnosis1. Culture

E. Treatment and Prevention1. Antibacterial - Penicillin and Tetracyclines2. Cleaning of wound, drainage of abscess3. Limiting contact to wild and domestic animals

-end-

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