MICROBIOLOGY- Bacterial Etiologic Agents of CAP

PARASITOLOGY Maria Elena R Sevidal-Saquilayan, MD, DPCP

LEVEL 2
October 18, 2019

OUTLINE - Enterobacteriaceae (Klebsiella pneumoniae) and

Pseudomonas spp.
ETIOLOGIC AGENTS OF COMMUNITY-ACQUIRED PNEUMONIA - Coxiella burnetti
(CAP) ................................................................................. 1
- Viruses:
HAEMOPHILUS INFLUENZAE ............................................... 1 o RSV
CHARACTERISTICS ................................................................... 1 o PIV
CULTURE .............................................................................. 1 o Human metapneumovirus
ANTIGENIC STRUCTURE.............................................................. 2
PATHOGENESIS ....................................................................... 2 o Influenza A and B
CLINICAL FINDINGS .................................................................. 2
PNEUMONIA ........................................................................... 2 HAEMOPHILUS INFLUENZAE
Clinical Setting ................................................................... 2
CHARACTERISTICS
Treatment ......................................................................... 2
Prevention ........................................................................ 2 - Small, Gram(-) pleomorphic bacteria
- Change its shape (pleomorphic)
MYCOPLASMA SP. ............................................................... 2
CHARACTERISTICS: .................................................................. 3 - Requires enriched media → blood containing
CULTURE & GROWTH CHARACTERISTICS............................................ 3 - Found on the mucous membranes of the upper respiratory
MYCOPLASMA PNEUMONIAE .................................................. 3 tract of humans
Characteristics ................................................................... 3
- Become opportunistic once the immune system of the host
Clinical Findings ................................................................. 4
Pathologic Findings ............................................................. 4 is compromised
Complications .................................................................... 4
Other Related Diseases ....................................................... 4
Diagnosis .......................................................................... 4
Laboratory Tests ................................................................ 4
Treatment ......................................................................... 4

CHLAMYDIA (CHLAMYDOPHILA ) PNEUMONIAE .................. 4

CHARACTERISTICS ................................................................... 4
5 MAJOR PHASES OF ITS REPLICATION ............................................. 5
ANTIGENS............................................................................. 5
Immunofluorescence........................................................... 5
GROWTH AND METABOLISM.......................................................... 5
CLINICAL FINDINGS .................................................................. 6
LABORATORY DIAGNOSIS ............................................................ 6
TREATMENT ........................................................................... 6

LEGIONELLA PNEUMOPHILA ............................................... 6

CHARACTERISTICS ................................................................... 6
CULTURE .............................................................................. 6
ANTIGENIC STRUCTURES ............................................................ 6
HISTORICAL PERSPECTIVE ........................................................... 7
PATHOGENESIS ....................................................................... 7
Fig 1. Haemophilus influenzae
CLINICAL FINDINGS .................................................................. 7
DISEASES CAUSED ................................................................... 7
Legionnaire’s Disease .......................................................... 7 - At 6-8 hours in rich medium → small, coccobacillary forms
Pontiac Fever ..................................................................... 7 - Later → longer rods, very pleomorphic
DIAGNOSIS ........................................................................... 7
LABORATORY TESTS.................................................................. 7
- Possess capsule (virulence factor) – in young cultures (6-
TREATMENT ........................................................................... 8 18 hours)
PREVENTION .......................................................................... 8 o Capsule antigen is used for serotyping
MORAXELLA CATARRHALIS ................................................. 8
CHARACTERISTICS ................................................................... 8 CULTURE
CAUSE PNEUMONIA IN: .............................................................. 8
- On IsoVitaleX-enriched Chocolate Agar:
DIAGNOSIS............................................................................ 8
TREATMENT ........................................................................... 8 o flat, grayish brown colonies
o Require the X (acts as Hemin) and V (NAD:
KLEBSIELLA PNEUMONIAE ................................................. 8 Nicotinamide-Adenine-Dinucleotide) factors in blood
CHARACTERISTIC ON CULTURE ...................................................... 8
CLINICAL FINDINGS .................................................................. 9 - Blood agar is couped so that a brown color appears
Complications .................................................................... 9 - Exposed in 80°C for the brown color to appear
Friedlander’s pneumonia...................................................... 9 - Does not cause hemolysis - γ hemolysis
TREATMENT ........................................................................... 9

ETIOLOGIC AGENTS OF COMMUNITY-ACQUIRED

PNEUMONIA (CAP)
- Streptococcus pneumoniae
- Haemophilus influenzae
o These 2 are the most common causes of CAP
- Moraxella catarrhalis
o Branhamella catarrhalis or Neisseria catarrhalis (old
name)
- Staphylococcus aures
- Legionella pneumophila

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- Chocolate agar – flat, grayish brown colonies after 24 hours

incubation
- Does not grow on sheep blood agar except around colonies
of Staphylococci → “satellite phenomenon”

ANTIGENIC STRUCTURE
1. Capsular polysaccharides (a-f)
- Strains (a-f) are based on the kind of capsule
- Type B – A polyribitol-ribose-phosphate (PRP) (Most
important)

2. Outer membrane proteins

- Lipooligosaccharides (endotoxins)

PATHOGENESIS
- Non-encapsulated H. influenza → normal flora
- Virulence factor → capsule

Fig 2. Mueller-Hinton Agar Disk Test
Haemophilus influenzae require both X & V factors to grow.
Haemophilus parainfluenzae requires V factor only for
growth. Haemophilus ducreyi requires only factor X without
need of factor V. We usually use Mueller Hinton Agar for the
disk test.
CLINICAL FINDINGS
- H. influenzae Type B (HIB)
o Causes meningitis in children (prior to the introduction
of vaccine (HIB vaccine);
o it is the main cause of meningitis in young children
between the age 5 months to 5 years
- Causes epiglottitis - (in infants; cherry red epiglottitis),
(Medical emergency. Can cause obstruction and respiratory
distress) and cause acute respiratory syndrome
- Septic arthritis (most common cause in infants)
- Causes bronchitis or pneumonia in adults

PNEUMONIA
Clinical Setting
- Chronic
- Associated with cardiopulmonary disease
- Follows upper respiratory tract infections
- Chest Radiograph/X-ray
o Lobar consolidation (Or even infiltrations)

Fig 3. H. influenzae on IsoVitaleX-enriched Chocolate Treatment

Agar - Ampicillin + Sulbactam (Beta-lactamase inhibitor)
- 3rd generation cephalosporins – Cefotaxime
- Given IV – for meningitis

Prevention
- Hib conjugate vaccine (H. influenzae type B)
- Given 3 doses (2,4,6 months) or
- 2 doses (2 and 4 months);
- Booster dose (between 12 & 15 months)

T/N: (12& 18 months sa Jawetz…)

MYCOPLASMA SP.
- Mollicutes (cell wall-free bacteria)
- 200 species; 16 are associated with human diseases; 4 are
of medical importance:
o M. pneumoniae
o M. genitalium
▪ closely related to M. pneumoniae
▪ has been associated with urethral and other
urogenital infections
o M. hominis
▪ sometimes causes postpartum fever
▪ has been found with other bacteria in uterine tube
infections

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o M. urealyticum
- Last 3 Mycoplasma species mentioned above cause non-
gonococcal urethritis and PID
o Ureaplasma urealyticum is a cause of non-gonococcal
urethritis in men and is associated with lung disease in
premature infants of low birth weight

CHARACTERISTICS:
- Smallest organisms that can be free in nature
- Highly pleomorphic (don’t have cell wall which makes them
change shape)
- Bounded by a three-layered cytoplasmic membrane that
contains sterol
o Makes it different from other bacteria because other
bacteria have no sterol in their cytoplasmic membrane
o Requires serum cholesterol in culture medium to produce
sterols for growth
- Completely resistant to penicillin Fig 5. Mycoplasma sp. colonies showing “fried egg
o Penicillin inhibits the cell wall bacteria and Mycoplasma appearance”.
is cell-wall-less
- Inhibited by tetracycline or erythromycin (macrolides)
MYCOPLASMA PNEUMONIAE
- Most important in Mycoplasma sp.
- Prominent cause of community-acquired pneumonia esp. in
persons 5-20 years of age
- Mode of transmission is person to person by means of
infected respiratory secretion
o Initiated by attachment of the tip (P1 adhesin) of the
organism to the receptor on the surface of respiratory
epithelial cells
- During infection remain extracellular
- Later may have the possibility to become intracellular
- Goes inside the infected host cell

Fig 4. Cellular structure of Mycoplasma sp.

CULTURE & GROWTH CHARACTERISTICS

- Grown on cell-free media that contain lipoprotein and sterol
- Form minute colonies → round, with granular surface and a
dark center typically buried in the agar
o “Fried egg appearance”
o Requires lipoprotein and sterol
- Require serum, glucose, or urea and yeast extract in the
culture medium Fig 6. Illustration of M. pneumoniae cell layer.

Characteristics
- Reside extracellularly in the respiratory & urogenital tracts
- Adherence: P1 pili
- Toxic metabolic products: peroxide & superoxide
- Immunopathogenesis: activate macrophages, stimulate
cytokine production

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Complications
- Uncommon
- Hemolytic anemia

Other Related Diseases

- Erythema multiforme (Hypersensitivity reaction)
- CNS involvement:
o Meningitis
o Meningoencephalitis
- Mono- and polyneuritis
o If peripheral nerves are involved
- Myocarditis, pericarditis
- Arthritis, Pancreatitis

Diagnosis
- Largely made on the basis of the clinical recognition of the
syndrome
o Fever and cough with normal findings but x-ray shows
extensive infiltrate

Laboratory Tests
- Slightly elevated WBC count
- Sputum Gram stain; PMNs and monocytes, no bacterial
pathogens
o To rule out other bacterial causes (S. pneumoniae)
- Culture: almost never done; hard to isolate
- Cold hemagglutinin: for group O human erythrocytes
(50% of untreated patients)
- Enzyme Immunoassay (EIA) – detect IgM and IgG
Fig 7. Diagram on how mycoplasma infection occurs. antibodies
- PCR assays of throat swabs specimens
Clinical Findings
- Incubation Period: 1-3 weeks (time of exposure to the Treatment
time of onset of symptoms) - Tetracycline, Macrolides, Fluoroquinolone
- Insidious onset: o Produce improvement but do not eradicate M.
o Malaise, fever, sore throat and cough (initially pneumoniae
nonproductive, later with blood streaked-sputum and CHLAMYDIA (CHLAMYDOPHILA ) PNEUMONIAE
chest pain) CHARACTERISTICS
- Causes atypical pneumonia (most common cause)
o Normal PE findings but with the x-ray, you will see
striking consolidation or extensive patchy infiltrates on
radiographs
o Other organisms that can cause atypical pneumonia:
Mycoplasma, Chlamydia, Legionella
- Resolution of pulmonary infiltrates occur in 1-4 weeks

Fig 9. An image of a cell infected with c. pneumoniae.

- Obligate intracellular parasites with a tropism for the

columnar epithelial cells
o Lack the mechanism for energy production so they
Fig 8. Chest X-ray of Patient with Mycoplasma cannot synthesize ATP, they need the mechanism from
pneumoniae the host cell
- Cell envelope - similar to that of Gram (-) bacteria
Pathologic Findings o Cell wall contains high lipid content
- Interstitial with peribronchial pneumonitis - Contain both DNA and RNA
- Necrotizing bronchiolitis o Differentiated from the virus that are intracellular
organisms
- Non-motile

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- Lack a mechanism for the production of energy 5. Release of EB from host cell, ready to infect another host
- Produce round, dense, intracytoplasmic inclusions that lack cell
glycogen
o Differs this from other species
- Humans are the only known hosts
- Resistant to sulphonamides
o Other species of chlamydia are susceptible

Fig 10. Image of C. pneumoniae on different stains.

Giemsa stain of Chlamydia inclusion bodies (purple Fig 12. Life cycle of Chlamydia pneumoniae
“caps” on epithelial cell).
ANTIGENS
- Only bacteria that exists in two distinct forms: - Group-specific antigens
o Heat-stable lipopolysaccharides with 2-ket0-3-
ELEMENTARY BODY RETICULATE BODY deoxyoctanoic acid as an immunodominant component
Small, dense, pear-shaped - Species-specific antigens
Larger, osmotically fragile
body o Mainly outer membrane proteins
The infectious form Metabolically active form
Attaches and enters into Form that divides by Immunofluorescence
target cells binary fission - Growth of Chlamydia spp. In McCoy cells stained with a
Contains equal amounts of Contains about 4 times fluorescein-labeled antibody against a C. trachomatis spp.
DNA and RNA RNA than DNA Antigen
Stains purple with Giemsa Stains blue with Giemsa - Intracytoplasmic inclusions of C. trachomatis stain bright
yellow-green

Fig 11. Cell stained with Giemsa revealing elementary

forms of chlamydia.

5 MAJOR PHASES OF ITS REPLICATION

1. Attachment and replication of the EB
- EB infects and penetrates the host cell

2. Reorganization of the metabolically inert EB into a

metabolically active form (Reticulate Body) Fig 13. Image of immunofluorescence staining for
Chlamydia.
3. Growth and division of the RB and production of many
progeny GROWTH AND METABOLISM
- RB multiplies - HL or Hep-2 cells (McCoy cells)
o Needs a cell for growth because they are obligate
4. Maturation of the non-infectious RB into infectious EB parasites
- Embryonated eggs

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CLINICAL FINDINGS
- Atypical pneumonia
o Clinically similar to M. pneumoniae but prodromal
symptoms last longer up to 2 weeks
o Usually affecting teenagers and young adults
o Chest radiograph: subsegmental infiltrate
▪ Less prominent than the infiltrate in M. pneumonia
o Consolidation is rare
o Common cause of atypical pneumonia with M.
pneumoniae
- Pharyngitis
- Sinusitis
- Otitis media

Fig 15. Legionella pneumophila

CULTURE
- BCYE media (buffered charcoal yeast extract) with alpha-
ketoglutarate and iron
- pH 6.9, T = 35°C, and 90% humidity
- Grow slowly (3 days incubation)
- Colonies: colorless to iridescent pink or blue and are
translucent and speckled
- Catalase (+), Oxidase (+)
- Hydrolyzes Hippurate
- Produces gelatinase and beta-lactamase
Fig 14. CXR showing infiltrates.
ANTIGENIC STRUCTURES
LABORATORY DIAGNOSIS
- 16 serogroups
- Culture - Serogroup 1 – cause of outbreak of Legionnaire’s
o HL of Hep-2 cells with cycloheximide (inhibits disease
metabolism of eukaryotic cells o Can also cause Pontiac fever
o Incubated at 35˚C for 3 days - (+) cross-reactive antigenicity among different species
- Serology: - Produces proteases, phosphatase, lipase, DNase, RNase,
o Microimmunofluorescence test metalloprotease
▪ Most sensitive method for the diagnosis of C. - Found in warm, moist environments, lakes, streams
pneumoniae infection o Aquatic setting that promotes bacterial growth (stagnant
water)
TREATMENT - Infection follows inhalation of bacterial from aerosols
- Tetracycline generated from contaminated air-conditioning systems and
- Macrolides shower heads
- Fluoroquinolone

LEGIONELLA PNEUMOPHILA
CHARACTERISTICS
- Fastidious (complex growth requirements)
- aerobic (require oxygen for growth, killed by the absence of
oxygen)
- Gram (-) pleomorphic bacteria, but stain poorly by Gram’s
method
o Basic fuchsin (0.1%) should be used as the
counterstain
- Do not occur as a commensal flora in man

Fig 16. Where Legionella sp. can be found

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HISTORICAL PERSPECTIVE o Steroid and other immunosuppressive agents

- July 1976: American Legion Convention in Philadelphia, o Cancer therapy
Pennsylvania ▪ Biologic agents as target, eg. Anti-TNF
- 2000 Legionnaires attended o Diabetes mellitus
- 184 Legionnaires develop pneumonia - If these occur w pneumonia, consider Legionella
- 29 Legionnaires and 5 others staying in the same hotel died pneumophila
of illness
DISEASES CAUSED

Legionnaire’s Disease
- More severe form
- High fever, chills, malaise, non-productive (prominent)
cough, hypoxia, diarrhea, and delirium (due to high fever)
- Chest x-rays:
o Reveal patchy, multilobular consolidation
- There may be leukocytosis, hyponatremia, hematuria, or
abnormal liver function test
- Antibiotics mandatory

Fig 17. Image of the legionnaires at the 1976

convention.

- Thermally heated bodies of water: hotels & hospitals

- Gain entry into cooling towers of large buildings and
evaporative condensers of:
o A/Cs, humidifiers, nebulizers
o Do not occur as a commensal flora in man

PATHOGENESIS
- Readily enters & grows within human alveolar macrophages
and monocytes
- Do not require opsonization by C3b / antibody to enter
macrophage
- Mip protein → virulence factor
Fig 19. CXR of a patient with Legionnaire’s disease.

Pontiac Fever
- Milder form (flu-like manifestation)
- Fever and chills, myalgia, malaise, and headache that
develop over 6-12 hours, dizziness, photophobia,
confusion
- Mild cough and sore throat
- Self-limited
- You may not give antibiotics

DIAGNOSIS
- Based on clinical features
- Exclusion of other causes of pneumonia by laboratory tests

LABORATORY TESTS
- Specimens
o Bronchial washings
o Pleural fluid
o Lung biopsy specimens
o Blood
Fig 18. Lifecycle of L. pneumophila. - Smears
o Bacteria not demonstrable in Gram-stained smears
CLINICAL FINDINGS - Direct fluorescent antibody test is diagnostic
- Silver stains
- Disease is highest in men > 55 years old
- Factors associated with high risk: o Used on tissue specimens
- PCR can also be done
o Smoking
o Chronic bronchitis - Legionella urinary antigen test
o May be used early in the course (specific for serogroup
o Emphysema
1)

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- Culture CAUSE PNEUMONIA IN:

o BCYE CM – of sputum or tissue specimens - Those w pre-existing lung disease
- Serologic tests - Elderly
- Those on corticosteroid
- Immunosuppressive therapy

DIAGNOSIS
- Chest radiograph
o Patchy infiltrates; occasional lobar consolidation

TREATMENT
- Trimethoprim-sulfamethoxazole (Cotrimoxazole) or
- Amoxicillin-clavulanate or
- 2nd or 3rd generation cephalosporin
o 1st generation – Gram (+)
o 2nd generation – Gram (+) and (-)
o 3rd generation – Gram (-)

KLEBSIELLA PNEUMONIAE
- Member of Enterobacteriaceae
- Gram (-) rods, non-motile
- With capsules – produce mucoid colonies
Fig 20. Direct fluorescent antibody stain showing L.
- Lactose fermenters
pneumophila.
- Produce urease
o Only lactose fermenter that produces urease
TREATMENT
- Tetracycline, Macrolides, Quinolones
- Prolonged therapy (may be up to 3 weeks)

PREVENTION
- Hyperchlorination and superheating of water
- Maintain and clean cooling powers
- Avoid conditions that allow water to stagnate
o Frequent flushing of unused water lines

MORAXELLA CATARRHALIS
CHARACTERISTICS Fig 22. Klebsiella pneumoniae
- Small gram (-) bacilli, coccobacilli, or cocci in pairs
- Oxidase (+)
- Non-motile, non-fermentative
- Produce DNase
- Produce butyrate esterase
o Basis for rapid fluorometric tests for identification
- Members of the normal microbiota of the upper respiratory
tract
- Often produce beta-lactamase
o Cause disease of immunocompromised patients

Fig 23. Capsule of K. pneumoniae.

CHARACTERISTIC ON CULTURE
- On Blood Agar (BA)
o Slimy appearance of the colonies

Fig 21. Microscopic image of M. catarrhalis.

Fig 23. K. pneumoniae on BA.

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- On MacConkey Agar (Mac)

o Red/pink colonies

Fig 24. K. pneumoniae on Mac. Fig 26. PA and Lateral view of CXR showing lobar
consolidation.
- On EMB
o Produces large, mucoid, pink to purple colonies with no Complications
metallic green sheen
- Cavitation and empyema/pus (like S. aureus [#1 cause of
empyema] and L. pneumophila pneumonia)

Friedlander’s pneumonia
- Severe form of CAP caused by K. pneumoniae with a
predilection for upper lobes of the lungs
- Liable to suppurate and form abscess
- Seen in patients with chronic debilitating illnesses or
alcoholism
- Alcoholic + currant jelly + infiltrates in upper lobe

Fig 25. K. pneumoniae on EMB

CLINICAL FINDINGS
- Causes pneumonia in:
o Persons on alcohol abuse
o Those with diabetes
mellitus
- Can be nosocomial (hospital-
acquired)
- “Currant jelly” sputum
(reddish jelly-like) Fig 27. Friedlanders’ pneumonia.
o Rusty sputum in S.
pneumoniae TREATMENT
o Alcoholic + currant jelly = - 3rd or 4th generation cephalosporin
Klebsiella pneumoniae - For severe infection: add Gentamicin or Tobramycin
- Chest radiograph
o Lobar consolidation (like
S. pneumoniae and H.
influenzae)

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