BACTERIAL INFECTION ON UPPER RESPIRATION TRACT

Ike Irmawati P.A, MSi Med Revised by eri dian Mikrobiologi FK Yarsi

Infections of the Respiratory tract

Most common entry point for infections Upper respiratory tract nose, nasal cavity, sinuses, mouth, throat (laring) Lower respiratory tract Trachea, bronchi, bronchioles, & alveoli in the lungs
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Upper & Lower Respiratory Tract Infection

Type microorganism: - restricted to surface - spread through body Two groups of microbes can distinguished : professional & secondary invaders
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Protective Mechanisms
Normal flora: Commensal organisms Limited to the upper tract Mostly Gram positive or anaeorbic Microbial antagonist (competition)

Protective Mechanisms
Clearance of particles and organisms from the respiratory tract For the upper respiratory tract : The mucociliary system in the nasopharynx The flushing action of saliva in the oropharynk

Bacterial Infections
Specific areas of the upper respiratory system can become infected to produce pharyngitis, laryngitis, tonsillitis, sinusitis, and epiglottis. These infections may be caused by several bacteria and viruses, often in combination.

Sore Throat
Clinical features: pharyngitis tonsillitis Causative organisms
mostly viral bacterial causes:
Streptococcus pyogenes
(Group A beta-haemolytic streptococci)

Less commonly: Corynebacterium diphtheriae, Group C and G beta-haemolytic streptococci, Arcanobacterium haemolyticum, Fusobacterium necrophorum, Haemophylus influenzae type B, Borrelia vincenti

Otitis Media & Sinusitis

Local spread of organisms from URT , e.g. Streptococcus pneumoniae Haemophilus influenzae viruses probably commonly involved Clinical features Fever, local pain, dizziness, deafness

SMEAR OF SPUTUM

In this smear of sputum there are large numbers of lancet-shaped gram-positive diplococci that represent pneumococcus and tangles of filamentous gram-positive organisms. In addition, however, in the background there are an even larger number of pleomorphic gram-negative bacilli that represent Haemophilus influenzae.

Acute Epiglottitis
Infective emergency Caused by Haemophilus influenzae capsular type B (Hib) Now very rare due to Hib vaccine program Can lead to acute respiratory obstruction
Diagnosis take blood cultures H. influenzae requires rich medium (e.g. lysed blood or chocolate agar) Requires X and V growth factors on nutrient agar Management Intravenous cefotaxime or ceftriaxone Isolate the patient Prevention Hib vaccine

Diphtheria

Acute toxin-mediated disease caused by Corynebacterium diphtheriae Gram-positive aerobic bacillus Incubation period 2-5 days Typically involves pharynx and tonsils leathery adherent membrane, which can cause respiratory obstruction Toxin effects Myocarditis Neuropathy

Streptococcal infection on Respiratory Tract

Streptococci
Characters of Streptococci
Gram positive cocci 1m in diameter Chains or pairs Usually capsulated Non motile Non spore forming Facultative anaerobes Catalase negative Of these organism considered in this chapter: S.pyogenes, S.pneumoniae, S.viridans

Classification of Streptococci
Streptococci can be classified according to:
Oxygen requirements
Anaerobic (Peptostreptococcus) Aerobic or facultative anaerobic (Streptococcus)

Serology (Lanciefield Classification)

Hemolysis on Blood Agar (BA)

Serology: Lanciefield Classification

Streptococci Lanciefield classification

Group A S. pyogenes

Group B S. agalactiae

Group C S. equisimitis

Group D Enterococcus

Other groups (E-U)

Streptococci classified into many groups from A-K & H-V One or more species per group Classification based on C- carbohydrate antigen of cell wall Groupable streptococci A, B and D (more frequent) C, G and F (Less frequent) Non-groupable streptococci S. pneumoniae (pneumonia) viridans streptococci e.g. S. mutans Causing dental carries

Classification of Streptococci Based on Hemolysis on Blood Agar

Hemolysis on BA -hemolysis
Partial hemolysis Green discoloration around the colonies This results from the oxidation of hemoglobin to methemoglobin by hydrogen peroxide secreted by the bacteria e.g. non-groupable streptococci (S. pneumoniae & S. viridans)

-hemolysis
Complete hemolysis Clear zone of hemolysis around the colonies This hemolysis occurs due to an enzyme produced by bacteria called streptolysin. Streptolysin interacts with cholesterol in the cellular membrane resulting in deterioration of this protective celluler membrane e.g. Group A & B (S. pyogenes & S. agalactiae)

-hemolysis
No lysis e.g. Group D (Enterococcus spp)

Hemolysis patterns on blood agar

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Hemolysis on Blood agar

-hemolysis

-hemolysis

-hemolysis

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Pathogenesis and Spectrum of Disease

Organism
S.pyogenes

Virulence Factors
Protein F & lipoteichoic acid mediates epithelial cell attachment &adhesion M protein as antiphagositic The M protein has many antigenic varieties and thus, different strain of S.pyogenes cause repeat infections
Hyaluronic acid capsule, which acts to camouflage the bacteria Produce enzyme and hemolysins-contribute tissue invasion and destructions,i.e: streptolysin O,S,streptokinase,DNAse,&hyaluronidase Streptococcal Pyrogenic exotoxins that stimulate macrophages and helper T cells to release cytokines

Spectrum of respiratory infections

Pharyngitis,pneumonia,scarlet fever,Streptococcal toxic shock syndrome Rheumatic fever

S.pneumoniae

Polysaccharide capsule inhibit phagocitosis Pneumolysin Production of extracellular complex pollysaccharide-enhanced attachment to host cellular surface,such as cardiac endothel

pneumonia

S.viridans

Subacute bacterial endocarditis

Pathogenesis S.pyogenes
Pathogenesis of group A streptococci

Adherence to the epithelial cells;

>10 adhesion molecules

invasion into the epithelial cells; mediated by M protein and F protein important for persistent infections and invasion into deep tissues avoiding opsonization and phagocytosis; M protein, M-like proteins, and C5a peptidase producing enzymes and toxins

Complication that result from S. pyogenes infection (poststreptococcal disease)

Rheumatic fever: Life threatening inflammatory disease that leads to damage of heart valves muscle Rheumatic fever: most commonly preceded by infection of the respiratory tract. Inflammation of heart (pancarditis), joints, blood vessels, and subcutaneous tissue. Results from cross reactivity of anti-M protein Ab and the human heart tissue. This disease can be reactivated by recurrent streptococcal infections Deposition of antibody-streptococcal Ag complexes in kidneys results in damagee to glomeruli---------------------->glomerulonephritis

S. pneumoniae
Morphology and Physiology

Gram-positive lancet-shaped diplococci for typical organisms.

alpha-hemolytic Growth is enhanced by 5-10% CO2.

*Quellung reaction (for rapid identification or typing of the bacteria)

These organism may harmlessly inhabit the upper respiratory tract When it gain access to the lungs by aspiration -acute suppurative pneumoniae When it accesses the bloodstream & meninges-acute,supurative often lifethreatening infections

Contd S. pneumoniae
Pathogenesis and Immunity Pneumococci produce disease through their ability to multiply in the tissues (invasiveness). Virulence factors: capsule, cell wall polysaccharide, phosphorylcholine, pneumolysin, IgA protease, etc. 40-70% of humans are at sometimes carrier of virulent pneumococci. Major host defense mechanisms: ciliated cells of respiratory tract and spleen. The normal respiratory tract has natural resistance to the pneumococcus. Loss of natural resistance may be due to:

1. Abnormalities of the respiratory tract (e.g. viral RT infections).

2. Alcohol or drug intoxication; abnormal circulatory dynamics. 3. Patients undergone renal transplant; chronic renal diseases. 4. Malnutrition, general debility, sickle cell anemia, hyposplenism or splenectomy, nephrosis or complement deficiency. 5. Young children and the elderly.

S. pneumoniae virulence factors

S. pneumoniae
Clinical diseases
Pneumococcal pneumonia develops when the bacteria multiply rapidly in the alveolar space after aspiration. The affected area is generally localized in the lower lobes of the lungs (lobar pneumonia). Children and the elderly can have a more generalized bronchopneumonia. Resolution occurs when specific anticapsular antibodies develop. Sudden onset with fever, chills and sharp chest pain. Bloody, rusty sputum. Empyema (mostly caused by type 3) is a rare but significant complication. Complications caused by spreading of pneumococci to other organs: sinusitis, meningitis, endocarditis, septic arthritis, middle ear infection.

Streptococcus viridans
Alpha hemolytic or nonhemolytic Nongrupable S.viridans colonizes in the oropharynx,GI tract,urinary tract, and rareli on skin surface. Generally considered to be of low virulence Production of extracellular complex polysacarides (such as glucans&dextrans) enhance attachment to host cell surface,such as cardiac endothelial cells & tooth surface in the case of dental caries Disease: subacut bacterial endocarditis (particularly patients with previosly damaged heart valves) Intra abdominal infection caries dentis

Diagnosis of streptococcal disease

Clinical diagnosis Depend on signs&symtoms Streptococcal disease Laboratory diagnosis
Antigen detection test Antibody detection methode Culture Identification

Laboratory diagnosis S. pyogenes

Antigen detection methode: Antigen detection tests: commercial kits for rapid detection of group A streptococcal antigen from throat swabs, using latex agglutination,coagglutination & Elisa tecnology. Latex agglutination able to detect Capsuler pollysacharide antigen of pneumococcus Serological test/antibody detection methode: ASO titration for respiratory infections.

Anti-DNase B and antihyaluronidase titration for skin infections.

Antistreptokinase; anti-M type-specific antibodies. Culture: Specimens are cultured on blood agar plates in air.

Identification

Laboratory diagnosis S. pneumoniae

Examination of sputum Stained smears of sputum: a rapid diagnosis. Quellung test with multivalent anticapsular antibodies. Culture

Specimen: sputum, aspirates from sinus or middle ear, CSF.

cultured on blood agar plate in 5-10% CO2. Identification: bile solubility, optochin sensitivity, etc. for differentiation from other a-hemolytic streptococci. Additional biochemical, serologic or molecular diagnostic tests for a definitive identification. Antigen detection: detect capsular polysaccharide in body fluids.

Outline of differentiation between GramPositive cocci

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Identification of Gram-positive cocci

None

Differentiation between -hemolytic Streptococci

Hemolysis Bacitracin sensitivity Susceptible Resistant CAMP test Negative Positive

S. pyogenes S. agalactiae

Differentiation between -hemolytic Streptococci

Hemolysis Optochin sensitivity
Sensitive ( 14 mm)
Resistant (13 mm)

Bile Inulin solubility Fermentation

Soluble
Insoluble

S. pneumoniae
S. viridans

Not ferment
Ferment

Prevention and Control

Most streptococci are normal flora of the human body Source of S. pyogenes and S. agalactiae is a person harboring these organisms (carrier). Control: 1. Prompt eradication of streptococci from early infections.

2. Prophylactic antibiotic treatment for rheumatic fever patients.

3. Eradication of S. pyogenes from carriers.

4. Dust control, ventilation, air filtration, UV irradiation and aerosol mists are of doubtful efficacy.
5. Intrapartum penicillin to mother at risk of giving birth to an infant with invasive group B disease.

Haemophilus

Characteristics of Haemophilus
Small, pleomorphic gram-negative coccobacilli, non motile (+) cytochrome oxidase Growth in culture requires exogenous hemin (oxidized ferroprotoporphyrin) (X factor) and/or nicotinamide adenine dinucleotide (NAD) (V factor)

Haemophilus: Natural Habitats

Normal inhabitant of the upper respiratory, gastrointestinal, and genital tracts of humans except Haemophilus ducreyi (not normal microbial flora)

Haemophilus species of clinical importance

1. H. influenzae
-type

b is an important human pathogen transmitted pathogen (chancroid)

2. H. ducreyi
-sexually

3. Other Haemophilus are normal flora

- H. parainfluenzae

- H. aphrophilus - H. aegyptius

Haemophilus: Modes of Infection

Encapsulated strains of Haemophilus influenzae associated with invasive infection caused by person-to-person spread of H. influenzae due to inhalation of infectious respiratory droplets Type b H. influenzae was most commonly associated with disease prior to conjugate vaccine but prevalence has declined with advent of vaccination

Haemophilus: Types of Infectious Disease

Encapsulated (types a-f) strains of H. influenzae produce invasive infection (pneumonia, meningitis, epiglottitis, and bacteremia) Unencapsulated (non-typeable) strains of H. influenzae cause otitis media in children, and lower respiratory tract infections (acute tracheobronchitis, pneumonia) in children and adults

Haemophilus: Species Identification

Preliminary findings: small faintly-staining (with safranin) gram-negative coccobacillary to filamentous rods that grow on chocolate agar but not sheep blood agar (except H. aphrophilus that with passage in culture grows on both) X and V factor requirements determined using X, V, and XV factor impregnated paper strips on MuellerHinton agar

Haemophilus influenzae

Differentiation of Species
Hemolysis Growth Factor
X V

H. influenzae H. aegyptius H. ducreyi H. parainfluenzae H. aphrophilus

+ -

+ + + -

+ + + -

Haemophilus influenzae
Aerobic gram-negative bacteria Polysaccharide capsule Six different serotypes (a-f) of polysaccharide capsule 95% of invasive disease caused by type b

Haemophilus influenzae type b Clinical Features*

Epiglottitis 17% Meningitis 50% Pneumonia 15%

Osteomyelitis 2% Arthritis 8% Cellulitis 6%

Bacteremia 2%

*prevaccination era

Haemophilus influenzae type b Epidemiology

Reservoir Human Asymptomatic carriers Respiratory droplets

Transmission

Haemophilus influenzae type b Pathogenesis

Organism colonizes nasopharynx In some persons organism invades bloodstream and cause infection at distant site Antecedent upper respiratory tract infection may be a contributing factor

Pathogenic Mechanisms
H. influenzae
Antiphagocytic polysaccharide capsule is the major pathogenesis factor

Lipopolysaccharide lipid A component from the cell wall (major role in non capsule strains)
All virulent strains produce neuraminidase and an IgA protease No exotoxins

Pathogenesis Host Factors

Hib conjugate vaccine (Poliribitol phosphate ( PRP) capsule) The Hib conjugate vaccine does not protect against nontypeable strains Persons at risk for invasive H influenzae disease
Asplenia Immunocompromised

Basis for the H.influenzae type B vaccine

The polysaccharide capsule of H.influenzae type B is and represent its major virulence antiphagocitic Factor. The capsule contain ribose,ribitol,and phosphate,known collectively as polyribitol phosphate (PRP). Phagocytosis and complement-mediated activity are stimulated in the presence of antibodies directed at the H.influenzae type B capsule. H.influenzae type B vaccine contain PRP antigens conjugated to specific protein carriers.

H. influenzae serotype b:
diagnosis and treatment timeline
Incubation Signs or symptoms Infectiousness Lab Specimens Prophylaxis

Exposure Incubation period unknown (~ 2-4d)

Onset* Sudden Onset Management of sequelae

Infectious while organisms are present, or until 24-48h of antimicrobial therapy

Immunize contacts aged <5y

Specimens from sterile site, for culture (cerebrospinal fluid, blood, pus, middle ear fluid). Gram Stain is presumptive

Antimicrobial prophylaxis

*Invasive disease includes meningitis, epiglottitis, pneumonia, septic arthritis, and cellulitis (less commonly osteomyelitis and pericarditis). Modified from Michigan Health Department http://www.michigan.gov/documents/mdch/2Hflu_Rev2008_231415_7.pdf

Haemophilus influenzae
IsoVitaleX-enriched chocolate agar
Requires 2 erythrocyte factors for growth: X (hemin) and V (NAD). X & V factors are released following lysis of red blood cells

5% CO2 enhances growth

Satellite Phenomenon H. influenzae

Grows near S. aureus on blood agar: satellite phenomenon.

Satellite phenomenon

S.aureus

Public Health AspectsH. influenzae

Typing based on capsule polysaccharide a f Polyribose-ribitol phosphate (PRP) capsule (type b) Nonencapsulated (nontypeable) organisms are part of normal flora of the respiratory tract

95% of invasive disease caused by type b

Polysaccharide Conjugate Vaccines

Stimulates T-dependent immunity Enhanced antibody production, especially in young children Repeat doses elicit booster response

Corynebacterium

Corynebacterium
Classification Corynebacterium diphtheriae and diphtheroids (look like C.diphtheriae) Some are saprophytic Some produce disease in animals C. diphtheriae is the most important pathogen in the group. - Other Corynebacterium are part of the normal flora of the skin and URT.

Corynebacterium
Are called diphtheroids and may occasionally cause disease, particularly in immunocompromised individuals.
C. ulcerans toxigenic strains may produce a disease similar to, but less severe than diphtheria. J-K Group commonly cause infections in those with underlying disease.
Diseases include bacteremia, meningitis, peritonitis, wound infections, etc. It is becoming more and more of a problem.

C. pseudotuberculosis found in those with exposure to animals.

Can cause pneumonia or lymphadenitis. Produces a different exotoxin than C. diphtheriae.

Biological Features
Aerobic, Gram+, Noncapsulated, rods,
arrangement=palisade or Chinese letters

Gray-black colonies on tellurite medium Metachromatic granules (Babes ernest)

Chinese-letter morphology in Gram stain

Diphtheroids Gram stain

Arrangement of C. diphtheria

Corynebacterium
Loefflers agar slant contains serum and egg that enhance the formation of metachromatic granules (polymerized polyphosphoric acid) in C. diphtheriae. Also called Babes-Ernst granules. They are visualized by staining with methylene blue.

Corynebacterium
3 morphological types of C. diphtheriae are found on tellurite containing media: Mitis black colonies with a gray periphery Gravis large, gray colonies Intermedius small, dull gray to black. All produce an immunologically identical toxin. Incubation -35-370 C for 24 hours. They prefer a pH of 7.8-8.0 for good growth. They require access to oxygen (poor An O2 growth). Biochemistry Catalase + Nonmotile C. ulcerans is urease +, C. diphtheriae is -, and C. pseudotuberculosis is usually +

Corynebacterium
Virulence factors C. diphtheriae For C. diphtherias to cause diphtheria an exotoxin must be produced. Is a heat-labile polypeptide produced during lysogeny of a phage that carries the "tox gene. Alkaline pH of 7.8- 8.0, aerobic conditions, and a low environmental iron level are essential for toxin production (occurs late in the growth of the organism). The toxin inhibits protein synthesis by ADP-ribosylating elongation factor 2.

Corynebacterium
Trypsin cleaves the toxin into 2 fragments, A and B, that are linked together by a disulfide bridge. Fragment B is required for toxin binding to tissue cells and fragment A contains the toxic activity. One molecule of toxin can inhibit 90% of the protein synthesis in a cell. C. ulcerans and C. pseudotuberculosis sometimes make a diphtheria-like toxin.

Diphtheria Toxin (DT)

Cleaved to yield A/B fragment, joined by S-S bond - A (catalytic domain) - B (transmembrane and receptor binding domains) Receptor: heparin-binding epidermal growth factor - rich on cardiac cells and nerve cells Toxin diffuses throughout body via blood - Cardiac, neurologic complications - Heart/respiratory damage, paralysis

C. diphtheria toxin
Toxin enters through receptor mediated endocytosis Acidification of endocytic vesicle allows A to dissociate from B A enters cycoplasm

Corynebacterium
To prove that an isolate can cause diphtheria, one must demonstrate toxin production. This is most often done on an Elek plate: The organism is streaked on a plate containing low iron. A filter strip containing anti-toxin antibody is placed perpendicular to the streak of the organism. Diffusion of the antibody into the medium and secretion of the toxin into the medium occur. At the zone of equivalence, a precipitate will form.

Elek plate

Pathogenesis of diphtheria

Early stages: Sore throat. Low fever. Swollen neck glands. Late stages: Airway obstruction and breathing difficulty. Shock

Corynebacterium
Clinical Significance (C. diphtheria) Is normally found in the throats of healthy carriers. The organism infects only man and it has a limited capacity to invade. Diphtheria - Disease usually starts as a local infection of the mucous membranes causing a membranous pharyngitis Local toxin effects result in degeneration of epithelial cells. Inflammation, edema, and production of a pseudomembrane composed of fibrin clots, leukocytes, and dead epithelial cells and microorganisms occurs in the throat.

Diphtheria - pseudomembrane
This may obstruct the airway and result in suffocation.

Corynebacterium
The more dangerous effects occur when the toxin becomes systemic and attacks the heart (heart failure), peripheral nerves (paralysis), and the adrenal glands (hypofunction). Cutaneous diphtheria More common in tropical and subtropical areas. Necrotic lesions with occasional formation of a local pseudomembrane occur.

DIAGNOSIS
Clinical: Muscle weakness, edema and a pseudomembranous material in the upper respiratory tract characterizes diphtheria. Laboratory: Tellurite media is the agar of choice for isolation of Corynebacteria, which produce jet black colonies

Corynebacterium
Shick skin test like the Dick test in that it tests for circulating antibody to the toxin by injecting a small amount of toxin intradermally and observing for a local erythematous and necrotic reaction. If this occurs it indicates that the person has no anti-toxin antibodies and is, therefore, susceptible to diphtheria.

Control
Sanitary: Reduce carrier rate by use
of vaccine.

Immunological: A vaccine (DPT)

prepared from an alkaline formaldehyde inactivated toxin (i.e. toxoid) is required. Passive immunization with antitoxin can be used for patients. Chemotherapeutic: Penicillin, erythromycin or gentamicin are drugs of choice.

Protection C.diphtheriae
Can be established through both active and passive immunity. Active immunity consist of a toxoid administered in the form of the DPT vaccine Passive immunity is established by administering diphtheria antitoxin. Antimicrobial therapy (erythromycin) can be used to effectively treat patient with clinical bacteria

Moraxella

Characteristics of Moraxella
Gram-negative diplococci with adjacent sides flattened Frequently appear as intracellular gramnegative diplococci within polymorphonuclear neutrophils

Characteristics of Moraxella
Growth Moraxella catarrhalis occurs on both sheep blood and chocolate agar Moraxella catarrhalis capnophilic (optimal growth with 3-7% CO2)

Characteristics of Moraxella
Moraxella rather than Branhamella accepted taxonomically as the genus designation for M. catarrhalis (family Moraxellaceae) Even though M. catarrhalis not a member of the family Neisseriaceae, morphologic and biochemical similarity to Neisseria allows clinical laboratory identification of M. catarrhalis with Neisseria species

Moraxella catarrhalis: Natural Habitats Present in the upper respiratory tract of 1.5-5.4% of healthy individuals, more commonly in children (50.8%) and elderly adults (26.5%)

Moraxella catarrhalis: Modes of Infection

Oropharyngeal endogenous strains spread into normally sterile regions of the tracheobronchial tree, the middle ear, and sinuses

Moraxella catarrhalis: Types of Infectious Disease

Acute purulent exacerbation of chronic bronchitis Causes 10-15% of episodes of otitis media and sinusitis Rarely associated with systemic infection (endocarditis, meningitits)

Moraxella catarrhalis: Identification

Pink coloration of colonies on chocolate agar not apparent on blood agar No acid from glucose, maltose, sucrose, fructose, or lactose (asaccharolytic) Produces DNase Possesses the enzyme butyrate esterase

M. catarrhalis

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Selected Biochemical Reactions for Identification of Neisseria and Moraxella catarrhalis1

Glu Mal Lac Suc DNa BE N. gon + N. men + + N. lac + + + M. cat + + 1Glu=glucose, Mal=maltose, Lac=lactose, Suc=sucrose, DNa=DNase, BE=butyrate esterase (indoxyl butyrate substrate), N. gon=N. gonorrhoeae, N. men=N. meningitidis, N. lac=N. lactamica, M. cat=Moraxella catarrhalis

Fusobacterium species
anaerobic, nonspore-forming, gramnegative bacilli Human infection usually results from F. necrophorum subspecies funduliforme, but infections with other species including F. nucleatum, F.gonidiaformans, F. naviforme, F.mortiferum, and F. varium have been reported

 Fusobacterium infections are most common in adolescents and young adults, but infections, including fatal cases of Lemierre disease, have been reported in infants and young children Diagnosis: anaerobic blood culture