Microbiology Lab 5

Bacterial Upper Respiratory infections

(URI)

What is considered the “upper respiratory tract”?

• By strict definition the upper respiratory tract includes the nasal passages, the middle ear, the nasopharynx, and the oropharynx. It also includes the large lymph nodes located in the oropharynx, the most important of which are called the pharyngeal tonsils.

What is the normal flora of the mouth comprised of?

• A wide variety of organisms comprise the normal flora of the mouth and upper respiratory tract. Like the skin and other body surfaces, the mucosa of the upper respiratory tract is heavily colonized with obligate anaerobes including Bacterioides, Fusobacteria, and anaerobic streptococci.

• Large numbers of facultative streptococci are alos found in the upper respiratory tract, especially in the mouth.

• These streptococci are of several species and may be hemolytic, non-hemolytic, or green (“alpha-hemolytic”) streptococci of the viridans group, Streptococcus pneumoniae is also found in the oropharynx in a high percentage of normal people, but most of these isolates are “rough”, indicating that they lack type-specific polysaccharide capsules.

What are the most numerous gram-negative cocci in the upper respiratory tract?

• The most numerous Gram-negative cocci are the Neisseria, and many different species of Neisseria are normally present.

• Members of the genus Haemophilus, including H.influenzae are also part of the normal oropharyngeal flora.

What are some important facts about Streptococcus species?

• Cultures of Group A beta hemolytic Streptococcus (Strep. Pyogenes) grow on sheep blood agar.

• Group A beta hemolytic streptococci produce hemolysins, which are capable of lysing red blood cells and converting hemoglobin to colorless products.

• This phenomenon produces a clear zone around each colony on the blood agar through which a newspaper can be read.

• This is termed beta hemolysis. • “Alpha” hemolysis, a result of hydrogen peroxide production, is incomplete hemolysis;

the zone around the colony is green and the RBC’s are intact.• Another diagnostic feature of Strep. Pyogens is its sensitivity to very low

concentrations of bacitracin.• This feature allows a rapid presumptive identification of beta hemolytic Streptococcus

as Group A.• A disk containing a very small amount of bacitracin (“A” disk – for Group A

streptococci) is placed over a heavily inoculated area of the plate. If there is marked inhibition of growth about the disk and surviving colonies away from the disk show beta hemolysis, one presumes that the organism is a Group A beta hemolytic Streptococcus.

• Grouping by means of serologic techniques can be performed to confirm this impression, as other streptococci, e.g. Groups B, C and G may also exhibit beta-hemolysis.

• It is important to remember that nearly everyone has many different strains of streptococci in their oropharynx and posterior nasopharynx. Thus, it is impossible to make even a tentative diagnosis of a “Strep. Throat” on the basis of a Gram Stain.

What are some important facts about Streptococcus Pyogenes (concise)?

• Obligate human parasite.• Spread person to person by respiratory secretions.• Causes strep throat, scarlet fever, impetigo, cellulitis, rheumatic fever (indirectly), and

acute glomerular nephritis (indirectly).• Catalase negative• An easy test to differnetiate S. pyogenes from other beta-hemolytic streptococci is the

bacitracin test.• S. pyogenes is killed by bacitracin (A disk), others are resistant.• Treated with PCN.• M protein is the most important virulence factor of S. pyogenes. M protein extends

from cell envelope as fimbriae. It has both conserved and variable regions. Structurally, it resembles a human protein since it has a coiled-coil alpha-helical structure. It prevents phagocytosis by preventing complement opsonization.

• Antibody to a specific M terminus provudes immunity to that M type.• Specific M protein types are associated with pharyngitis or skin infections. Likewise,

specific M protein types are associated with rheumatic fever and acute glomerular nephritis.

• The rash of scarlet fever is caused by SPE (s. pyogenes exotoxins), aka Erythrogenic Toxin.

What are some important facts about Streptococcus Pneumoniae?

• Alpha-hemolytic Gram positive cocci.

• Lancet shaped, non-motile.

• Inhibited by optochin (P disk) and bile.

• Catalase negative (Used to differentiate between strep & staph)

• Does not contain cytochromes, like other streptococci species, so it can only ferment (no ETC). This also means that it is intrinsically resistant to aminoglycosides since they use the ETC to enter the cell.

• The antiphagocytic capsule is the main virulence factor of S. pneumoniae. This capsule has many serotypes.

• Very common cause of pneumonia.

• Most common cause of meningitis.

• Splenectomy, alcoholism and hypogammaglobulinemia increased the risk of disease due to s. pneumoniae.

• There is a vaccine against S. pneumoniae that contains the 20 most common capsular antigens. Recently, a conjugated vaccine was released for children.

What are some important facts about Viridans Streptococci?

• Alpha-hemolytic

• Resistant to Optochin

• Normal flora

• Cause dental carries

• Ferment glucose to lactic acid, and long-term exposure to the acid destroys the enamel of the teeth.

• May frequently cause endocarditis.

What are some important facts about Bordetella pertusis?

• Strick aerobe.

• Classically, Bordetella pertussis does not invade. Rather it colonized the respiratory cilia and produces toxins which diffuse into the systemic circulation.

• Causes a biphasic disease primarily in children <12 y/o. Adults can be carriers or have mild symptoms. A patient will be immune upon recovery from natural disease.

• Very fastidious. Forms colonies that look like bisected pearls in 3-6 days on initial isolation, Bordet-Genjou agar is used for the culture. This agar is made of a potato infusion, glucose, glycerol, peptones, and 15-30% blood.

• It can also be diagnosed with a fluorescent antibody test from a nasopharyngeal swab or by using PCR.

• Important virulence factors include: filamentous hemagglutinin, pertacin, pertussis toxin, adenylate cylcase/hemolysin and pili. Bvg controls the expressions of many of its unlinked virulence genes.

What are some important facts about Haemophilus influenzae?

• Grow on Chocolate agar.

• Very pleomorphic.

• Requires X (hematin) and V (NAD) for growth.

• Capsule is the major virulence factor.

• Only capsule type B causes invasive diseases such as pneumonia, meningitis, and epiglotitis. Capsule is comprised of polyribosyl ribitol phosphate (PRP).

• Other virulence factors are IgA protease, LPS, etc.

• Unencapsulated forms commonly colonize the nasopharynx without causing disease.

• The HiB vaccine conjugates PRP to a protein so that children as young as 2 months can raise a response to PRP using T dependent immunity.

• PCN resistance is becoming a problem.

How do you distinguish Strep pneumoniae from viridans?

• These streptococci are clearly differentiated from Strep pyogenes by type of hemolysis, but hemolysis does not distinguish Strep pneumoniae from viridans.

• Fortunately, Strep pneumoniae is extremely sensitive to bile and similar detergents, which activate autolytic enzymes, resulting in rapid bacterial lysis, whereas viridans is not so sensitive.

• This is shown by a bile solubility test, which, when positive, is indicated by clearing of the bacterial suspension (indicative of pneumococci).

• The optochin disk provides an efficient alternative to bile solubility suitable for bacterial growth on solid media.

• The optochin disk (P disk, “p” for pneumoniae) is placed over the heavily inoculated areas of each of the plates.

• One should note the hemolytic pattern and the sensitivities to bile and optochin to differentiate between streptococcus species.

What are some important facts about Haemophilus influenzae?

• Haemophilus influenzae cause otitis media, sinusitis and bronchitis.• Haemophilus influenzae type B, for which there is an effective

vaccine, also causes invasive infections such as bacteremia and meningitis.

• Grown on Chocolate agar. (recall haemophilus = blood loving)• X factor = Hematin (hemin)• V factor = NAD

What are some important facts about Mycoplasma pneumoniae?

• The mycoplasma most pathogenic for humans is called Mycoplasma pneumoniae. Mycoplasma do not have a rigid cell wall and cannot be grown on standard laboratory media. Several characteristics of mycoplasma pneumoniae distinguish this species from other saprophytic mycoplasmas.

• Colonial morphology, rapid hemolysis of guinea pig red blood cells, and hemadsorption are all diagnositc features of M. pneumoniae.

• Hemolysis is observed around colonies that are growing in agar medium containing guinea pig red blood cells.

• Hemadsorption is observed by adding a solution of guinea pig red blood cells to colonies of myocplasma pneumoniae growing on the surface of nutrient agar.

• Colonies are much smaller than bacterial colonies.

What antibiotics are mycoplasma sensitive to?

• In contrast to gram-positive bacteria with rigid cell walls, which are sensitive to antibiotics that inhibit cell wall synthesis, mycoplasma pneumonia is sensitive only to those antibiotics that directly inhibit protein or DNA synthesis or attack plasma membranes.

What is “convalescent serum” and how can you use it to tell if someone has been infected with mycoplasma pneumoniae?

• Several types of antibodies are formed in response to mycoplasma pneumoniae infection. While normal human serum should aid in the growth of Mycoplasma pneumoniae, serum from a patient with a recent infection should inhibit the growth.

• “Convalescent serum” is from someone who has recovered from an infection and will have antibodies to the mycoplasma. Mycoplasma will have little or no growth on these convalescent plates.

What does Bordetella pertussis cause?

• Bordetella pertussis is the cause of whooping cough, classically in infants and children but now also seen in adolescents and adults.

• It is a tiny, coccobacillary aerobic, gram-negative rod.• Pertussis occurs in epidemics and more localized outbreaks.• Infection begins in the nasopharynx and extends into the tracheo-

bronchial tree, where the effects of pertussis toxin paralyze the ciliary function of the epithelium.

• Diagnosis can be made by visualizing bacteria in secretions using fluorescein-labeled antibody (rapid results), or by culture (takes 7-10 days); however neither is sensitive enough to rule out the diagnosis.

• Due to its sensitivity and rapid turn-around time, PCR is an attractive alternative (in combination with culture). Limitations of PCR for the diagnosis of pertussis include false positive results and lack of a standardized and FDA-approved test.

How do they look? Let’s see…

H. flu requires X (hematin) and V (NAD)Brown is NOT GROWTH! Look in light:

No growth

Growth

H. Flu

Grows onChocolate agar

Bordetella pertussis on Bordet-Genou“bisected pearls”

Streptococci -- comparisons on BAP

S. pneumoniae

S. pyogenes S. viridans

P = optochinA = bacitracin

• Case1a: A 3 month old boy was brought into the emergency department for a cough and difficulty breathing. His mother states that she had noticed the cough for 10 days with increasing severity to the point where it was now difficult for the infant to catch his breath and it interfered with eating. She frequently noted prolonged coughing spells with occasional emesis. The infant’s 4 year old was not ill; however both parents had URI symptoms with a mild cough about 2 weeks ago. The boy had missed his 2 month immunizations due to a fever at the time.

• Vital signs: HR 145, RR 50, T 99.6• General: moderate respiratory distress, accessory muscle use, small left subconjunctival hemorrhage• CV: tachycardic, regular, 2/6 systolic murmur LLSB without radiation• Chest: clear on auscultation• Skin: no rash• CXR was normal• CBC was notable for a WBC of 17,000 with 75% lymphocytes.

• Case1b: A 58 year old male presents to his primary care doctor with a non-productive cough of about 2 weeks duration. The cough is worse at night and interferes with his sleep. Otherwise he feels well; he denies fever, chills, shortness of breath or chest pain. He has diabetes mellitus, hypercholesterolemia, and hypertension. He is not aware of any sick contacts and has not traveled recently. His medications include lisinopril, atorvastatin, and metformin.

• VS: HR 82, BP 146/87, RR 18, T98.9• CV: regular rate and rhythm without murmur• Chest: clear to auscultation and percussion• Extremities: no peripheral edema• CXR and laboratory studies are normal

• What is the most likely etiologic agent (assuming both cases are due to the same organism)? How is it spread? Bordetella pertussis. Spread by person to person by respiratory droplets.

• Describe the three clinical stages of this disease in its classic form.– Catarrhal phase, there is colonizaton of the respiratory epithelium causing local damage.– Paroxysmal phase, there is systemic intoxication with pertussis toxin.

• Symptoms include intense coughing, vomiting and convulsions.

– Third stage???

• If the same organism is responsible for both presentations – aside from age, what might account for the different presentations?

– May have been vaccinated….(???)

• Case2: 62 year old male with chronic obstructive pulmonary disease (COPD) (FEV1 = 1.3L) presented to the Emergency department with 3 days of increasing shortness of breath and dyspnea on exertion. He had a cough productive of yellowish-green sputum but no chest pain. The cough was increased both in severity and sputum production from his baseline. He denied fevers, chills, or sick contacts. He had increased the use of his inhalers without improvement.

• He is on 1-2L per minute of oxygen via nasal canula at home.• Thin male in moderate respiratory distress, pursed lip breathing. Unable to speak in complete sentences.• HR 115, BP 137/92, RR 30, T 99.3• O2-sat 85% on 2L oxygen, decreases to 80% with any activity.• CV: tachycardic with distant heart sounds. No murmur.• Chest: decreased breath sounds throughout, scattered rhonchi. End-expiratoyr wheezes.• Ext: no edema.• CXR: hyperexpanded lungs, no infiltrate.• CBC: WBC 10.2 with normal differential• Sputum culture grown on a chocolate agar plate yields small, coccobacillary gram-negative rods; no growth was noted

on blood agar.

• Which bacteria are commonly associated with acute exacerbations of COPD/chronic bronchitis?

– Haemophilus influenzae and Strep. pneumoniae

• Based on the culture what is the most likely organism contributing to this exacerbation?

• Haemophilus influenzae.

• In addition to bacteria, what other infectious agents are associated with COPD exacerbations?

– Viruses: The viruses identified in one study were influenza A (7.3%), coronavirus OC43 (4.6%), rhinovirus (3.1%), influenza B (2.7%), and respiratory syncytial virus (2.3%).