Microbio Lec 9 - Hemophilus Bordetella Francis Ella, Brucel

Lecture 9: Small Gram Negative Bacilli, Section C I. HEMOPHILUS MORPHOLOGY  Pleomorhic, 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.

H. aphrophilus ANTIGENIC STRUCTURE 1. Capsular polysaccharide – 6 antigenic types (a-f) 2. Lipopolysaccharide 3. Outer member protein MAJOR VIRULENCE FACTORS 1. Polysaccharide capsule (PRP) polyribitol phosphate – induce natural immunity after infection or vaccination 2. Lipopolysaccharide lipid A – induces meningeal irritation 3. IgA, specific proteases – facilitate colonization of mucosal surfaces 4. Outer-membrane proteins and pili – poorly defined roles EPIDEMIOLOGY OF HAEMOPHILUS INFECTION • Disease/ Bacterial Factors

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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

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PHYSIOLOGY  CULTURE MEDIA 1. Chocolate agar (heated blood) to 80°C. Factor X and V released 2. Levinthal enriched agar - differentiating - capsulated from non-capsulated 3. Fildes agar 4. 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

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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.

HAEMOPHILUS INFLUENZA ON CHOCOLATE AGAR HAEMOPHILUS INFLUENZA ON CHOCOLATE AGAR (close-up view)

2. 3.

• IDENTIFICATION OF HAEMOPHILUS INFLUENZA BY X AND V STRIPS

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

children < 5 years old without protective antibodies against type-b capsular polysaccharide patients with depleted complement of after splenectomy elderly at greatest risk for pulmonary disease, particularly those with obstructive pulmonary disease or conditions predisposing to aspiration

Geography/ season H. influenzae infections occur worldwide H. ducreyi genital ulcers in Africa and Asia, less commonly in Europe and North America No seasonal incidence

LABORATORY DIAGNOSIS 1. Gram staining 2. 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 Characteristics and growth requirements of some Hemophilus species (X = heme; V = nicotinamide-adenine dinucleotide) Requires Species Hemolysis X V H. influenzae (H. aegyptius) + + H. parainfluenzae + H. ducreyi + H. haemolyticus + + + H. parahaemolyticus + +

TREATMENT A. ANTIMICROBIALS 1. Meningitis – Chloramphenicol/+ Ampicillin 2. Otitis media – Amoxycillin, TMP-SMX, Erythromycin, Cefaclor 3. Sinusitis – Ampicillin B. PASSIVE IMMUNOTHERAPY  adjunct to antibiotics – in treatment of severe infection PREVENTION

1.

Active immunization 1985 – Purified capsular polysacc vaccine protective for 2 year old * Conjugated vaccines – with diphtheria and Neisseria meningitides 2. Passive Immunization Children with congenital or acquired deficiencies 3. Chemoprophylaxis – Rifampicin – Controversial OTHER HEMOPHILUS SPECIES 1. 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 socioeconomic and hygienic condition indurated, painful, single or multiple lesions, developing in the genitals and perianal areas, accompanied by suppurative inguinal buboes. II. BORDETELLA 3 Species B. pertussis (man is only natural host B. parapertusis (man is only natural host) B. bronchiseptica

Disease Pertussis or whooping cough Milder form of whooping cough Bronchopulmonary disease in animals and humans

MacConkey Agar

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Size: 0.2 – 0.5 X um Strictly aerobic, gram negative coccobacilli nonmotile, highly communicable, multiply among cilia of epithelial cells Culture medium  modified Bordet-Gengou medium (potatoglycerol-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 variant 1. Serial passage on culture media 2. High level of magnesium in media

Duration

Symptoms

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Incubation

Catarrhal

Paroxysmal

7-10 days

1-2 weeks

2-4 weeks

None

Rhinorrhea, malaise, fever, sneezing, anorexia

Repetitive cough with whoops, vomiting, leokocytosis

Convalescent 3-4 weeks (or longer) 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.

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Differential Characteristics of Bordetella Species Bordatella species B. B. B. Characteristics pertussis parapertussis bronchiseptica Oxidase + + Urease + + Motility + Browning on: Mueller-Hinton Agar + Growth on: Sheep Agar

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CLINICAL PRESENTATION OF B. PERTUSSIS DISEASE

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B. bronchiseptica on 5% sheep blood agar

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Virulence Factors Associated with Bordetella pertussis Virulence Factors Biological Effects ADP-ribosylation of guanine nucleotidebinding proteins, Pertussis toxins lymphocytosis, hypoglycemia, mediates attachment of respiratory epithelium Impairment of leukocyte Adenylate cyclase toxin chemotaxis and killing, local edema Ciliastasis and then Tracheal cytotoxin extrusion of ciliated epithelial cells Vascular smooth muscle Dermonecrotic toxin contraction and ischemic necrosis Mediates attachment to Filamentous hemaglutinin ciliated epithelial cells, agglutinates erythrocytes Lipopolysaccharide Exotoxin activity

PHYSIOLOGY AND STRUCTURE

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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-

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cysteine clood agar (CGB+) or peptone cysteine agar (RCA) - greenish disc surrounds the colony but no true hemolysis occurs

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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

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Higher Magnification

Colonies of Brucella sp. on 5% sheep blood agar

Worldwide; in the United States most infections are in Arkansas, Missouri, and Oklahoma Disease most frequent during summer and winter

C. Manifestations of Tularemia Infections Type of disease (%) Ulceroglandular

75-85

Glandular

5-10

Typhoidal

5-15

Oculoglandular

1-2

Oropharyngeal

<1

Ulcers at the site of exposure and adenopathy of the draining lymph nodes Adenopathy but no ulcers Systemic signs but no adenopathy or ulcers Eye involvement Orapharyngeal involvement

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 Physiology and Structure

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.

Characteristics

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 negative E. Modes of Control 1) Streptomycin (drug of choice), gentamicin 2) Avoid reservoirs and vectors infection(e.g., rabbits, ticks) 3) Live, attenuated vaccines reduce severity of disease 4) Lab personnel: use of gloves and biohazard hood when handling specimens

A.

Gram

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 stain of Brucella bacill

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 -

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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 Species B. melitensi s B. abortus

Animal Reservoi r

Growth on dyes CO2 Basic Thioni require fuchsi n d n

Goats, sheep

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Cattle

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+

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Clinical disease Severe, acute disease with complications common Mild disease with

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suppurative complications uncommon Species

Animal Reservoi r

Growth on dyes CO2 Basic Thioni require fuchsi n d n

B. suis

Swine

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B. conis

Dogs

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Clinical disease Suppurative, destructive disease with chronic manifestations Mild disease with suppurative complications uncommon

B. Epidemiology of Yersinia infection • Disease/ Bacterial factors Y. pestis – plague Y. enterocolitica – enterocolitis, transfusion-related septicemia Y. psuedotuberculosis – enterocolitis Y. pestis – present in animal reservoir, fleas Other Yesinia – present in domestic animals (GI tract) and contaminated food products Numerous virulence factors •

Transmission

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Antibody Responses in untreated Brucellosis

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D. Modes of Control 1) Tetracycline combined with streptomycin or with gentamicin 2) Long-term therapy with high doses of trimethoprimsulfamethoxazole 3) Control of disease in animal reservoir 4) Avoidance of unpasteurized dairy products 5) 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

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Strain identification thru ability to reduce nitrates

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?

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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

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

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Y. pestis: communities with endemic plague and exposure to infected animals Y. enterocolitica: individuals eating contaminated food, recipients of contaminated blood products Geography/ Season

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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 Pathigenicity 1. Ca2+ dependency 2. V and W antigen 3. outer membrane proteins 4. envelope antigen 5. pesticin, coagulase and plasminogen activation 6. pigment binding and iron regulated surface proteins D. Clinical Syndromes 1. 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

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Chemotherapy – streptomycin, alternative drugs as tetracyclines and chloramphenicol Kanamycin appears as effective as streptomycin

F. Prevention and Control 1. 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 control 4. 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 determinants 1. Inv (invasion) gene 2. Ail (attachment-invasion locus) 3. V and W Ag 4. Gene for Ca2+ concentration and temperature 5. 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 Syndrome 1. Diarrhea, fever and abdominal pain lasting for as long as 1-2 weeks 2. Chronic form can develop and persist for months to one year 3. Involves the ileum, with enlargement of mesenteric lymph nodes. Can mimic acute appendicitis 4. 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. Diagnosis 1. Culture of organisms from uncontaminated samples 2. Widal type agglutination test E. Treatment 1. Aminoglycosides 2. Trimetoprim and Sulfamethoxazole 3. 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 Manifestations 1. Infection via bites or scratches 2. Superinfection of chronically diseased lung 3. Other foci of infection secondary to septicemia D. Laboratory Diagnosis 1. Culture E. Treatment and Prevention 1. Antibacterial - Penicillin and Tetracyclines 2. Cleaning of wound, drainage of abscess 3. Limiting contact to wild and domestic animals

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