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 INFECTIOUS DISEASE

BACTERIOLOGY IMMUNOLOGY MYCOLOGY PARASITOLOGY VIROLOGY

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BACTERIOLOGY - CHAPTER  THIRTEEN 

STREPTOCOCCUS PNEUMONIAE  and Staphylococci 

Dr Alvin Fox

   

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Logo image © Jeffrey Nelson, Rush University, Chicago, Illinois  and The MicrobeLibrary

Reading: Murray, 6th Edition Chapters 21 and 22

KEYWORDS
S. pneumoniae
Diplococcus
Pneumococcus
Autolysin 
Bile solubility test
Optochin susceptibility
Capsule
Quellung reaction
Staphylococcus aureus
Staphylococcus epidermidis
Coagulase positive or Coagulase negative
Alpha, beta, gamma and delta cytotoxins
Leucocidin
Lipase
Exfoliatin
Enterotoxins
Toxic shock syndrome
Toxic shock toxin
Protein A



strep-pneu2.jpg (30090 bytes) Figure 1A  Streptococcus pneumoniae in spinal fluid. FA stain (digitally colorized). CDC/Dr. M.S. Mitchell 

 

STREPTOCOCCUS PNEUMONIAE 

S. pneumoniae (figure 1) is a leading cause of pneumonia in all ages (particularly the young and old), often after "damage" to the upper respiratory tract (e.g. following viral infection). It also causes middle ear infections (otitis media). The organism often spreads causing bacteremia and meningitis. S. pneumoniae is α hemolytic and there is no group antigen.

Direct Gram staining or detection of capsular antigen in sputum can be diagnostic. The organism grows well on sheep blood agar.

Autolysin

Pneumococci are identified by solubility in bile. An autolysin (peptidoglycan-degrading enzyme) is released by bile from the cell membrane and binds to a choline-containing teichoic acid attached to the peptidoglycan. The autolysin then digests the bacterial cell wall resulting in lysis of the cell. If the cells are grown in ethanolamine instead of choline, ethanolamine is incorporated into the teichoic acid. The autolysin then cannot lyse the cell wall. Understanding how the autolysin works has led to the suggestion that antibiotics (including penicillin) work together with the autolysin in killing of pneumococci in vivo.

The organisms are also identified by susceptibility to optochin (ethyl hydrocupreine) (figure 2)

Capsule 

This is highly prominent in virulent strains (figure 1c) and its carbohydrate antigens vary greatly in structure among strains. The capsule is anti-phagocytic and immunization is primarily against the capsule. Capsular vaccines are available for susceptible individuals; immunity is serotype-specific. Using appropriate type-specific antisera, the capsule on isolated bacteria can be "fixed" and becomes visible microscopically (the Quellung reaction) which is useful in microbial identification.

The organism also produces pneumolysin that degrades red blood cells under anaerobic conditions (observed as alpha hemolysis).

Complement activation by teichoic acid may explain the attraction of large numbers of inflammatory cells to the focal site of infection.

Most strains of S. pneumoniae are susceptible to penicillin. However, resistance is quite common.

 

strep-pneu.jpg (34733 bytes) Figure 1B  Scanning Electron Micrograph of Streptococcus pneumoniae. CDC/Dr. Richard Facklam  rrf2@cdc.gov  Spneumo.jpg (85081 bytes) Figure 1C Encapsulated Streptococcus pneumoniae   © Gloria J. Delisle and Lewis Tomalty, Queens University, Kingston, Ontario and The MicrobeLibrary

A spneu-opt.jpg (13412 bytes) smit-opt.jpg (17246 bytes) Figure 2  IIt is difficult to distinguish normal alpha streptococci found in the mouth from the pathogenic Streptococcus pneumoniae. Both are alpha-hemolytic on blood agar and so must be distinguished using the "P" disk (optochin). 
S. pneumoniae (A) is sensitive while S. mitis (B) is resistant 
© Pat Johnson, Palm Beach Community College, Lake Worth Florida

 


  Figure 3  Staphylococcus aureus - MRSA resistant coccoid prokaryote (dividing); causes food poisoning, toxic shock syndrome and skin and wound infections (scalded skin syndrome, scarlet fever, erysipelas, impetigo, etc.)  
© Dennis Kunkel Microscopy, Inc.  Used with permission

staph2.gif (14186 bytes)
Figure 4  Staphylococcus aureus (Gram-positive)  © Copyright Dr Linda M Stannard, 1996.  Used with permission

staph-orange.jpg (478200 bytes)  Figure 5  Staphyllococcus aureus - Acridine-orange leucocyte cytospin test  © Bristol Biomedical Image Archive. Used with permission

staph-impetigo.jpg (451762 bytes) Figure 6 Staphylococcal Infection: Impetigo © Bristol Biomedical Image Archive. Used with permission

cdc-rely.jpg (45994 bytes)  Figure 7 Box of Rely tampons. Associated with outbreak of toxic shock syndrome. CDC 

 staph-epi.jpg (60125 bytes) Figure 8
S. epidermidis, the most common cause of blood stream infections in patients with IVCs
© Nancy Khardori and Mahmoud Yassien, Southern Illinois University School of Medicine, Springfield, Illinois and The MicrobeLibrary

 

STAPHYLOCOCCI

Staphylococci are facultative anaerobes. They are Gram positive, occur in grape like-clusters and are catalase positive. They are major components of the normal flora of skin and nose in all people.

Staphylococcus aureus (figure 3 and 4)

S. aureus is one of the commoner causes of opportunistic infections in the hospital and community, including pneumonia, osteomyelitis, septic arthritis, bacteremia, endocarditis, abscesses/boils and other skin infections (figure 6). 

Pathogenesis

Food poisoning

S. aureus  produces a number of toxins, of which the enterotoxins (A, B, C and D) cause food poisoning. About a third to a half of  S. aureus strains produce enterotoxins which are heat stable and thus survive cooking (boiling for 30minutes). They are also resistant to proteolysis by intestinal proteases.

The food becomes contaminated with the organism from human contact, grows and produces enterotoxin. The organism does not "infect" the patient on ingestion of contaminated food; rather the pre-existing toxin causes the symptoms which include vomiting, nausea, diarrhea (watery and non-bloody, leading to dehydration) and abdominal pain. Fever is not observed. Because only the toxin is involved, onset of symptoms occurs within a few hours and recovery occurs within a day. Antibiotic treatment is not indicated because the bacteria are not directly involved in causing the symptoms (and may, anyway, have been killed by cooking).

Enterotoxins are superantigens that lead to cytokine production, T cell activation, neutrophil infiltration with loss of small intestine brush border cells. The release of inflammatory mediators may be the cause of the characteristic S. aureus food poisoning-associated vomiting.

Enterocolitis

The symptoms of enterocolitis are somewhat similar to food poisoning (watery diarrhea and abdominal pain) but also include fever. They are also produced by enterotoxin A and leukotoxin. The cause is the treatment of patients with broad spectrum antibiotics that allow S. aureus (which infects almost everyone) to grow in the intestine in preference to the normal bacterial flora. The bacteria can be detected in fecal samples.

Toxic shock syndrome

Toxic shock syndrome is caused by infection with strains of S. aureus that produces toxic shock syndrome toxin. It may be associated with a wound in which the bacteria multiply rapidly but became particularly prominent to the public in the 1980's when S.aureus infection was found to cause the toxic shock syndrome that was seen after the use of certain tampons such as "Rely" (figure 7). The bacteria were able to divide rapidly within the tampon; they do not disseminate but remain in the vagina. However, the toxin does disseminate and is responsible for the clinical features. This syndrome includes fever, macular erythrematous rash, desquamation (all over the body), vomiting and diarrhea. Toxic shock syndrome toxin has the properties of a superantigen, resulting in the production of cytokines, vascular leak and cell toxicity. This results in hypervolemic shock and death as a result of multi-organ failure. Before the cause of  toxic shock syndrome was discovered, the mortality rate was high but now is around 5%. There can be recurrent disease if the patient is not treated with the appropriate antibiotic.

Toxic shock syndrome toxin is involved in most menstruation-associated toxic shock syndrome. Enterotoxin B is involved in many non-menstruation associated cases of toxic shock syndrome.

Scalded skin syndrome (Ritter disease, pemphigus neonatorum) and bullous impetigo

A minority of S. aureus strains produce exfoliative toxins (A and B) and either toxin can cause scalded skin syndrome or bullous impetigo in babies and young children but rarely in adults. These toxins are serine proteases that can digest, among other proteins, some of the proteins found in desmosomes, the structures that link epithelial cells together. For example, the desmosomal protein called desmoglein is digested between the cells of the stratum granulosum epidermis. The process often resolves as the result of the formation of protective neutralizing antibodies. Exfoliative toxins are also superantigens.

Bullous impetigo is a mild form of S. aureus disease that usually occurs in newborn infants and young children. It is manifested by large, flaccid bullae and attributed to S. aureus strains belonging to phage group II capable of producing exfoliative toxins A and B that separate the stratum corneum from the rest of the epidermis. The more common and milder form of the disease―representing about 10% of all cases of impetigo―differs from non-bullous impetigo in that the vesicles enlarge into flaccid bullae before rupturing . The exposed skin surface is at first moist and red, resembling a small burn. A thin, light-brown, “varnish-like” crust then develops. Unlike the situation with scalded skin syndrome, bacteria can be cultured from the fluid of the bullae and Nikolsky's sign is absent.

The more severe form of disease with greater skin involvement caused by the same staphylococcal strains is known as the staphylococcal scalded skin syndrome. This also usually affects younger children. The disease starts with local peri-oral erythema that spreads over the whole body and progresses to widespread, flaccid bullae that rupture causing exfoliation of the skin that resembles an extensive third-degree burn. There are no organisms that can be cultured from the fluid of the bullae, indicating that the bullae are caused by the toxin and not the bacteria themselves. Before the bullae form, slight pressure on the apparently normal epidermis may separate it at the basal layer. It may be rubbed off when pressed with a sliding motion. This is Nikolsky's sign.

This form of the disease can occur in epidemic form in nurseries, where it is known as pemphigus neonatorum or Ritter’s disease. Fever and other systemic symptoms are usually absent in the more localized forms of the disease but are invariably present in patients with the staphylococcal scalded skin syndrome.

Localized bullous impetigo is self-limited due to the formation of neutralizing anti-toxin antibodies, and this is usually also the case with staphylococcal scalded skin syndrome. However, the latter carries a significant mortality rate (5%) that results from secondary bacterial infections of the areas where the skin surface has been lost. Staphylococcal scalded skin syndrome in adults is rare, and is usually associated with immunosuppression or kidney disease. In this case mortality can be as high as half of the patients.

Cytotoxins

As noted above, S. aureus causes a number of different disease entities associated with production of certain exotoxins. In addition to these "disease-specific" exotoxins, other cell lytic exotoxins (alpha, beta, gamma and delta toxins and leucocidins) may be produced. These are also called cytotoxins because they cause cytolysis as a result of plasma membrane damage. This leads to tissue destruction as a result of lysosomal enzyme release.

Alpha toxin

This singler polypeptide toxin interacts directly with the plasma membrane of many cells, embedding itself in the lipid bilayer and forming pores that allow ions to pass into and out of the cell. In particular, potassium ions are lost and sodium and calcium enter the cell. This leads to osmoticlysis. Alpha toxin is made by most S. aureus strains.

Beta toxin

Beta toxin also damages cell membranes by degrading specific lipids, sphingomyelin and lysophosptidyl choline. The toxin is a sphingomyelase C and is also a single polypeptide that is made by most S. aureus strains. It appears that the degree of toxicity depends on the concentrations of these lipids in the cell, both of which are found primarily in the outer monolayer of the plasma membrane bilayer.

Gamma toxins and P-V leukocidin

Gamma toxins and Panton-Valentine leukocidin consist of two polypeptide chains, an S chain and an F chain, which together form pores in the plasma membranes of susceptible cells. So far, three S chains and two F chains have been found which can combine to form a number of different toxins that are cytolytic to neutrophils and macrophages. The gamma toxins are also hemolytic whereas P-V leukocidin is not. The gamma toxins are also made by most S. aureus strains whereas P-V leukocidin is made by only a minority of strains. It has been particularly associated with virulent MRSA infections.

Delta toxin

This is a small protein that is cytotoxic to many cells. It may act like a detergent, damaging cell membrane bilayers resulting in cytolysis.

 

Other diseases caused by S. aureus

Respiratory disease

Aspiration pneumonia can result from entry of oral secretions into the lungs. The bacteria can cause local abscesses and infiltrates. The disease is found in the very young, the very old and patients with pulmonary disease. There can also be spread of blood-borne organisms to the lungs, causing hematogenous pneumonia. People with MRSA can get necrotizing pneumonia which has a very high fatality rate.

Empyemia is an accumulation of pus in a cavity of the body such as the lungs and is sometimes seen in pneumonia patients. Many of these cases are the result of S. aureus infections.

Bacteremia

S. aureus is found on the skin of most people and can enter the body in wounds; however, many cases are nosocomial and result from surgery or catheter use. The bacteria may disseminate throughout the body.

Endocarditis

Endocarditis is an inflammation of the endocardium (the inner layer of the heart) and usually involves the heart valves (native or prosthetic valves). S. aureus-associated endocarditis can have a high mortality rate.

Urinary tract infections

Complicated urinary tract infections occur in specific clinical settings. Renal abscess can result from hematogenous seeding of the renal cortex (most often due to S. aureus) or from ascending infection leading to severe pyelonephritis (most often due to gram-negative rods).

Dissemination to other parts of the body

S. aureus bacteremia can disseminate via the bloodstream to other parts of the body causing disease. Such sites include bone giving rise to S. aureus osteomyelitis resulting in pain, fever and sometimes a Brodie abscess and septic arthitis.

Skin disease

Folliculitis

Folliculitis, by which is meant pyoderma involving the hair follicles and apocrine glands, affects nearly everyone at one time or another but is usually self-limited. Occasionally, folliculitis evolves into larger lesions known as furuncles and carbuncles.
S. aureus is the usual cause of folliculitis in non-immunocompromised patients, the infection probably arising from prior nasal colonization by this bacterium.

Furuncles, carbuncles and skin abscesses

The familiar furuncle or “boil” is thought to arise from folliculitis. The term furunculosis refers to multiple boils or to frequent recurrences. Carbuncles are more extensive and difficult-to-treat lesions that often require surgical intervention. Skin abscesses, although similar to carbuncles histologically, are usually deeper infections that do not originate in hair follicles.

S. aureus is the usual cause of both furuncles and carbuncles, and is also the sole or predominant pathogen in about 50% of skin abscesses. Predisposing factors to recurrent furuncles (furunculosis) include obesity, corticosteroid therapy, disorders of neutrophil function, and possibly diabetes mellitus. Immunoglobulin levels are usually normal in patients with furunculosis (low IgM levels have been demonstrated in some patients but this is of uncertain significance and, in contrast to IgG deficiency, replacement therapy is impractical). Most patients with recurrent furuncles have no obvious predisposing factors other than being nasal carriers of S. aureus nasal carriers. Outbreaks of furunculosis have been described in families, athletic teams, and in village residents who took steam baths together. Skin abscesses can result from minor trauma, injecting drug use (the practice of subcutaneous and intramuscular injection is known as “skin popping”), or bacteremia. Congenital immunodeficiency syndromes such as the hyperimmunoglobulin E-recurrent infection syndrome (Job’s syndrome) are sometimes present in patients with recurrent skin abscesses. Rarely, skin abscesses are self-inflicted (factitious abscess), in which case Gram’s stain and culture may reveal “mouth flora” bacteria.

For more information see: Infectious disease - skin and bone

Other secreted enzymes

S. aureus strains secrete a number of tissue-degrading enzymes that may result in tissue damage. These include  lipases, nucleases, hyaluronidase, coagulase and plasmin. One form of coagulase is bound to the S. aureus surface and converts fibrinogen to fibrin. This insoluble protein causes the bacteria to aggregate. The other coagulase is secreted and combines with coagulase-reacting factor in the serum resulting in the formation of staphylothrombin that, like normal thrombin, also forms insoluble fibrin. This may also be anti-phagocytic.

 

Protection against phagocytosis

In addition, to the toxins and enzymes that directly damage cells and tissues described above, S. aureus strains produce other proteins involved in pathogenesis. For example, these bacteria have two mechanisms that protect them against phagocytosis by polymorphonuclear leukocytes and other phagocytic cells.

  • Although the bacteria are opsonised by proteins in serum, the capsule and slime layer protect the cells against phagocytosis.

  • Protein A is found on the surfaces of most S. aureus strains. It binds to immunoglobulin G and complement, blocking Fc and complement receptors and is thus anti-phagocytic.

Identification

  • S. aureus is beta-hemolytic on sheep blood agar

  • Ferments mannitol (figure 9)

  • Is often golden pigmented (hence the name aureus)

  • Is coagulase-positive

  • Presence of protein A

In reference laboratories phage-typing is used.

 

 

 

Staphylococcus epidermidis

Staphylococcus epidermidis (figure 8) is a less common cause of opportunistic infections than S. aureus, but is still significant. It is a mediator of nosocomial infections (e.g. catheters, shunts, surgery [e.g. heart valves]). It is a major component of the skin flora and thus commonly a contaminant of cultures.

Identification

  • Staphylococcus epidermidis is non-hemolytic on growth on sheep blood agar

  • Does not ferment mannitol (figure 9)

  • Is non-pigmented

  • Is coagulase-negative.

 

 

  Mannitol.jpg (41285 bytes) Figure 9  Two different species of Staphylococcus growing on mannitol salt agar (MSA).  MSA is selective because it contains 7.5% salt–a high salt concentration that promotes the growth of some organisms while discouraging the growth of others.  MSA is a differential medium because it contains the sugar mannitol and the pH indicator phenol red.  Organisms that can ferment mannitol produce acid by-products, causing a color change.  Phenol red is a cherry red color above pH 8.5, yellow-red from pH 6.9 to 8.5, and bright yellow at pH 6.9 or lower.  Although both Staphylococcus epidermidis and Staphylococcus aureus can tolerate the high salt content of MSA, only S. aureus can ferment mannitol, causing the phenol red in the medium to turn yellow. © Margaret (Peg) Johnson, Mesa Community College, Mesa, Arizona and The MicrobeLibrary

 

MOVIE
Catalase Test
  

Cultures of Staphylococcus aureus (left) and Streptococcus pyogenes (right) were grown on blood agar plates for 16 h at 37 degrees. A colony from each plate was placed on a glass slide. A drop of 3% hydrogen peroxide was placed on both organisms. The catalase- producing organism catalyzes the breakdown of H2O2 to oxygen and water. O2 is released as bubbles. The catalase test is used to differentiate Staphylococcus sp. from Streptococcus sp. Staphylococcus sp. are positive for catalase production. Streptococcus sp. are negative for catalase production.  © Neal R. Chamberlain, Department of Microbiology, Kirksville College of Osteopathic Medicine Kirksville, Missouri  and The MicrobeLibrary

 

 

 

Staphylococcus saprophyticus

This organism is a significant cause of urinary tract infections, usually in young women who are sexually active. Symptoms include dysuria and pyuria. It is also coagulase-negative and is not usually differentiated from S. epidermidis clinically.

 

 

Antibiotic therapy

Staphylococci (including both coagulase positive and coagulase negative organisms) can produce a phage-coded penicillinase that degrades beta lactam antibiotics. Some strains also have modified penicillin binding proteins. Thus, beta lactam antibiotics (including methicillin) are often ineffective.

Vancomycin is the drug of choice for treatment of methicillin-resistant S. aureus (MRSA); however, some strains of S. aureus have now developed resistance to this antibiotic as well.

 

 

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