Streptococcus Pneumoniae: Morphology, Identification, and Growth Features

University of Mustanisiriya College of Medicine Department of Microbiology Third stage STREPTOCOCCUS PNEUMONIAE Dr Ali Abdulwahid

The pneumococci (S. pneumoniae) Gram-positive diplococci Often lancet shaped or arranged in chains Possessing a capsule of polysaccharide that permits typing with specific antisera. Normal inhabitants of the upper respiratory tract of 5 40% of humans Can cause pneumonia, sinusitis, otitis, bronchitis, bacteremia, meningitis, and other infectious processes

The Morphology and identification Typical gram-positive, lancet-shaped diplococci In sputum or pus, single cocci or chains are also seen. With age, the organisms rapidly become gram negative and tend to lyse spontaneously. Sensitive to optochin, in contrast to viridans streptococci which are resistant to this antibiotic (differential feature) virulence for mice when injected intraperitoneally the capsule swelling test, or quellung reaction is a differential test to recognise this bacteria from other streptococci

Culture the organisms form small round colonies, at first dome shaped and later developing a central depression with an elevated rim. Other colonies may appear glistening because of capsular polysaccharide production. Fastidious (required enriched media such as blood agar and chocolate agar) Their growth is enhanced by 5 10% CO2 -hemolytic on blood agar.

Growth features The organism ferment the glucose along with a rapid production of lactic acid, which limits growth. Neutralization of broth cultures with alkali at intervals results in massive growth. Variation in capsule production: Production of capsule is not essential for growth on solid media, and the capsule production is lost after small number of subculture Isolates that produce capsule tend to form large and mucoid colonies Production of capsule can be returned again after injecting the bacteria into mice

Antigenic Structure

A. Component Structures The pneumococcal cell wall has peptidoglycan and teichoic acid, similar to other streptococci. The capsular polysaccharide is covalently bound to the peptidoglycan and to the cell wall polysaccharide. The bacteria classified to 91different serovar depending on the different in the fine structure of their capsular polysaccharide

B. Quellung Reaction This reaction depends on the reactivity of certain capsular polysaccharide with their specific antibodies (anti-capsular polysaccharide serum) or with polyvalent antiserum The polyvalent antiserum contains antibodies that were generated against all types of pneumococcal capsules Mixing the encapsulated pneumococci with the serum (specific or polyvalent) on a microscope slide leading to swelling of the capsule and agglutinating of the bacteria due to the cross linking of the antibodies with the capsule This test provides a rapid identification of the organism and also for typing them either in sputum or in cultures. The polyvalent antiserum is a good reagent for rapid microscopic determination of pneumococci is specimens such as sputum.

Pathogenesis

Pathogenesis Immunotypes of S. pneumoniae S. pneumonia serovar 1- 8 are responsible for around 75% of cases of pneumococcal pneumonia in adults Theses immunotypes are also responsible for more than 50% of mortality caused by pneumococcal bacteremia; Whereas in children, immunotypes 6, 14, 19, and 23 are the most frequent causes of infection

Main virulence factors 1. Capsule polysaccharide (C-Polysaccharide) The capsule is main virulence factor of this organism, which confers resistance or delay the ingestion by phagocytes. It also play a role in intiation the infection by facilitating the adhesion of the bacteria to host cells and cause inflammation Unencapsulated variants are not capable of causing disease 2. Pneumolysin: this toxine is presents in the cytoplasm of the bacteria and can be released after lysis of the cell The toxin results in forming pores in cell membranes of Human cells leading to lysis of the cells 3. Autolysin: is an autolysis enzymes involved in autolysis of bacteria and consequently release of pneumolysin and other components from within the cells 4. Pili: play a role in attachment and colonization of the pneumococci to the epithelial cell within the nasopharynx and lungs 5. immunoglobulin A1 (IgA1) protease

Frome carriage to disease state Around 40 70% of healthy individuals carrying the bacteria in their upper respiratory tracts asymptomatically The infection is usually arise from this normal flora the normal respiratory mucosa of such individuals must possess great natural resistance to the pneumococcus, and losing of such resistance lead to cause pneumococcal infection factors that lead to reduce such resistance and promote forming infection are the following: 1. Viral and other respiratory tract infections that damage surface cells; abnormal accumulations of mucus (eg, allergy), which protect pneumococci from phagocytosis; bronchial obstruction (eg, atelectasis); and respiratory tract injury caused by irritants disturbing its mucociliary function 2. Alcohol or drug intoxication, which depresses phagocytic activity, depresses the cough reflex, and facilitates aspiration of foreign material 3. Abnormal circulatory dynamics (eg, pulmonary congestion, heart failure) 4. Other mechanisms, such as malnutrition, general debility, sickle cell anemia, hyposplenism, nephrosis, or complement deficiency

Clinical findings Sudden onset of fever , chills, and sharp pleural pain Bloody or rusty coloured sputum In the early stage of the disease, the bacteraemia is presents in 10-20 % of cases . With antimicrobial therapy, the illness is usually terminated promptly; if drugs are given early, the development of consolidation is interrupted. Empyema (pus in the pleural space) is a significant complication and requires aspiration and drainage. From the respiratory tract, pneumococci may reach other sites. The sinuses and middle ear are most frequently involved. Infection sometimes extends from the mastoid to the meninges. Bacteremia from pneumonia has a triad of severe complications: meningitis, endocarditis, and septic arthritis

Diagnostic Laboratory Tests 1. Specimens Blood, for culturing CSF and sputum, for smear and culture. CSF and urine, for detection of C-polysaccharide by rapid immunochromatographic membrane assays. Specimens should be sent directly to the lab after collection because due to autolysis of pneumococci which is expected to happen and then affect the results of the tests.

Tests A. Smears for gram stain A Gram-stained film of rusty-red sputum shows typical organisms, many polymorphonuclear neutrophils, and many red blood cells. B. Capsule Swelling Tests Fresh emulsified sputum mixed with antiserum causes capsule swelling (the quellung reaction) for identification of pneumococci. C. Culture The culture is created by sputum cultured on blood agar and incubated in CO2 or a candle jar. A blood culture is also taken.

Treatment The bacteria is sensitive to many antibiotics, and the early treatment lead to a rapid recovery from the infection Penicillin G is the drug of choice for susceptible strains penicillin-resistant strains are currently spread worldwide Some of these strains are resistant to cefotaxime. Resistance to tetracycline, erythromycin, and fluoroquinolones ( which can be used as alternative to penicillin) also occurs. Pneumococci remain susceptible to vancomycin. Antibiotic susceptibility test should be performed for all infection to check the resistance profiles and chose the best antibiotic

Epidemiology. Diseases caused by S. pneumonia occur worldwide Infection caused by this bacteria are prevalence in young children, the elderly, and immunocompromised people. The organism also responsible for about 60% of all bacterial pneumonias. The infection developed only when the host of carriers lose their natural resistance for the bacteria ( as normal flora) due to several factors mentioned before. Although the organism can spread by air droplets in closed population, this is not play a significant role in spread of the infection and epidemic are rare. The healthy carriers is more important in disseminating pneumococci than a sick patient.

vaccination immunizing individuals with type-specific polysaccharides can probably provide 90% protection against bacteremic pneumonia. Currently there are two types of vaccines : 1. (PPSV-23) : A polysaccharide vaccine composed of purified capsules of 23 different serotypes of streptococcus pneomoniae This vaccine is recommended for adults between 19-64 years old who have chronic or immunosuppressing medical conditions. It is given routinely for individuals aged 65 years and older 2. The pneumococcal conjugate vaccine (PCV) : PCV-7 vaccine contains capsular polysaccharides of seven serotypes conjugated to diphtheria CRM197 protein. it was approved in 2000 and provided protection against serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F. It was recommended for all children ages 2 23 months to help prevent invasive infections and for selected children aged 24 59 months, Immunisation with this vaccine showed a significant reduction in invasive pneumococcal infections caused by those serotypes in the United States and other countries within 3 years of introduction PCV-13: (PCV-13) was recently approved. PCV-13 contains the polysaccharide conjugates of the serotypes found in the PCV-7 vaccine plus serotypes 1, 3, 5, 6A, 7F, and 19A. It is recommended for all children as a four-dose series at 2, 4, 6, and 12 15 months of age.

The references Reidle, S., Morse, S. A., Meitzner, T., and Miller, S. 2019. Jawetz, Melnick & Adelberg s Medical Microbiology , Twenty-Eight Edition. The McGraw-Hill education, Inc. USA

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