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Bacteriology lecture no. 2 Streptococcus *General characteristic 1-All species of this genius is catalase negative ,gram positive , cocci. 2-it is characteristically form pairs or chains during growth. 3- They are widely distributed in nature ,some are members of the normal human flora ,others are associated with important human diseases attributable in part to sensitization to them . 4-streptococci elaborate a variety of extracellular substances and enzymes . *Classification of Streptococci. The classification of streptococci into major categories has been based on a series of observations . 1- Colony morphology and hemolytic reactions on blood agar . 2- Serological specificity of the cell wall group – specific substances and other cell or capsular antigens . 3- Biochemical reactions and resistance to physical and chemical factors . 4- Ecologic features . a- *Hemolysis . Many streptococci are able to hemolysis RBC in vitro in varying degrees , complete disruption of RBC with cleaning of the blood around the bacterial growth is called β hemolysis, incomplete lysis of RBC with reduction of hemoglobin and the formation of green pigment is called α hemolysis , other streptococci are nonhemolytic sometimes called ƴ hemolysis . B- Group – specific substances ( Lancefield classification ) This CHO is contained in the cell wall of many streptococci and forms the basis of serologic grouping into Lancefield group A-H and K-u . The serologic specificity of the group – specific CHO is determined by an amino sugar . C – Capsular polysaccharides . The antigenic specificity of the capsular polysaccharides is used to classify S.pneomoniae into over 90 types and to type the group β streptococci ( S.agalactiae ) D – Biochemical reactions . Biochemical tests include sugar fermentation reactions tests for the presence of enzymes , and tests for susceptibility or resistance to certain chemical agents . *characteristics of medically important streptococci . Name Streptococcus pyogenes Groupspecific substance A Streptococcus agalactiae B Streptococcus dysgalactiae C,G Enterococcus faecalis D hemolysis Habitat Important criteria β Throat, skin None , α Colon Pharyngitis, impetigo rheumatic fever, glomerulonephritis toxic shock Neonatal sepsis and meningitis , bacteremia in adults Pharyngitis pyogenic infection – similar to group A streptococci Growth in presence Abdominal obsess UTI ƴ bite , hydrolyze , endocarditis esculin , growth ,in 6.5%NaCl ,PYR +ve Dental caries ,endocarditis ,abscess Large colonies , PYR test positive , inhibited by bacitracin β Female genital tract , lower Hippurate GI tract hydrolysis (Amppositive) β (human) Throat Large colonies α or none Viridans Usually not None,α streptococci typed or have many untypeable species Streptococcus pneumoniae None tab, Common and important diseases Α Mouth,throat,colon,female genital tract. nasopharynx Susceptible to Pneumonia, meningitis optochin ,colonies ,endocarditis, otitis soluble in bile, media, sinusitis quelling reaction positive *Streptococcus pyogenes - most Streptococci that contain the group A antigen are S. Pyogenes . - it is the main human pathogen associated with local or systemic invasion and post streptococcal immunologic disorders , - S Pyogenes typically produces large zones of β-hemolysis around colonies greater than 0.5 mm in diameter . - they are PYR – positive and usually are susceptible to bacitracin . *morphology and identification . A- Typical organisms . 1- Individual cocci are spherical or ovoid and are arranged in chains . 2- The members of the chain often base a striking diplococcal appearance , the lengths of the chain vary widely and are conditioned by environmental factors . 3- Streptococci are gram positive , however , as a culture ages and the bacteria die, they lose their gram positivity . 4- Most group A strains produce capsules composed of hyaluronic acid . This impede phagocytosis . 5- The capsule binds to hyaluronic – acid – binding protein , CD44 present on human epithelial cell ,Binding induces disruption of intercellular junctions allowing m.o. , to remain extracellular of intercellular junctions allowing m.o. to remain extracellular as they penetrate the epithelium . B- culture :1- Most streptococci grow in solid media as discoid colonies usually 1-2 mm in diameter . 2- It is typically β- hemolytic . C- Growth characteristics , 1- Energy is obtained principally from the utilization of glucose with lactic acid as the end product . 2- Growth of streptococci tends to be poor on solid media or in broth unless enriched with blood or tissue fluids . 3- Most pathogenic hemolytic streptococci grow best at 37C0 . 4- Most streptococci are facultative anaerobes and can grow under anaerobic conditions . *Antigenic structure :- A- M protein - This substance is a major virulence factor of group A S.pyogenes. M protein appears as a hair like projection of the streptococcal cell wall . when M protein is present, the streptococci are virulent, and in the absence of M type – specific antibodies , they are able to resist phagocytosis by polymorphonuclear leucocytes . S. Pyogenes that lack m protein are not virulent . - There are many , types of M protein may reach to 150 types and this explain why a person can’t develop immunity against Streptococcal pyogenic infection . - It appears that m protein and perhaps other streptococcal cell wall antigens have important role in the pathogenesis of rheumatic fever . - A component of the cell wall of selected M types induces antibodies that react with cardiac muscle tissue . B- T substances . this antigen has no relationship to virulence of streptococci unlike m protein , T substance is acid labile and heat – labile . C- Nucleoproteins . Extraction of streptococci with weak alkali yield mixtures of proteins and other substances of little serologic specificity , called P substances , which probably make up most of the streptococcal cell body . *Toxins and Enzymes . More than 20 extracellular products that are antigenic are elaborated by S. Pyogenes , including the following . A- Streptokinase ( fibrinolysin )it is produced by many strains of group A –βhemolytic streptococci , it transforms the plasminogen of human plasma into plasmin , an active proteolytic enzyme that digest , fibrin and other proteins , it has been given intravenously for treatment of pulmonary emboli, coronary artery , and venous thrombosis . B- Streptodornase or streptococcal deoxyribon nuclease , which depolymerizes DNA . purulent exudates owe their viscosity largely to deoxyribonucleorprotein , so this enzyme help to liquefy exudates and facilitate removal of pus and necrotic tissue , antimicrobial drugs thus gain better access , and infected surfaces recover more quickly . C- Hyluronidase : it splits hyaluronic acid , an important component of the grand substance of connective times . it aids in spreading infecting microorganisms . (spreading factor) it is antigenic and specific for each bacterial or tissue source . D- Pyrogenic exotoxins ( Erythrogenic Toxin ) These toxins are elaborated by S.pyogenes . There are 3 antigenic ally distinct Streptococcal pyrogenic exotoxins A,B and C. The streptococcal pyrogenic exotoxins have been associated with streptococcal toxic shock syndrome and scarlet fever , and most case is associated with type A , some rare cases may be attributed to type C while the role of type C is unclear . The pyrogenic exotoxins act as superantigens , which stimulate T cells by binding to the class 2 major histocompatibility complex in the VB region of the T cell receptor and the T cell release cytokines that mediate shock and tissue injury . E- Diphophopyridine Nucleotides . This enzyme is elaborated into the environment by some streptococci ,and this substance may be related to the organism’s ability to kill leukocytes . F- Hemolysins : The β- hemolytic group A S Pyogenes elaborates two hemolysin streptolysin O , it is hemolytically active in the reduced state and rapidly inactivated in the presence of oxygen , it is responsible for some of the hemolysis seen in blood agar , and it combines with antistreptolysin O (ASO) , an antibody that appears in humans following infection with any streptococci that produce streptolysin O , an ASO serum titer in excess of 160 – 200 units is considered abnormally high . - Streptolysin S .Is the agent responsible for the hemolytic zones around streptococcal colonies growing on the surface of blood agar plates , it is not antigenic and elaborated in the presence of serum . *pathogenesis and clinical findings :A variety of distinct disease processes are associated with S Pyogenes infections , the infection can be divided into several categories . A- Diseases attributable to invasion by S. Pyogenes ,β. Hemolytic Group A streptococci , the portal of entry determine the principal clinical picture . 1- Erysipelas :- if the portal of entry is the skin , erysipelas results , with massive brawny edema and a rapidly advancing margin of infection . 2- Cellulitis :- streptococcal cellulitis is an acute rapidly spreading infection of the skin and subcutaneous tissues , it fallows infection associated with mild trauma , burns , wounds or surgical incisions . 3- Necrotizing fasciitis (streptococcal gangrene ) there is extensive and very rapidly spreading necrosis of the skin and subcutaneous tissue this is infection of subcutaneous tissues and fascia . 4- Puerperal fever :- if the streptococcal enter the uterus after delivery, puerperal fever develop . 5- Bacteremia sepsis :- infection of traumatic or surgical wands with streptococci results in bacteremia , which rapidly can be fatal . B- Diseases attributable to local infection with S. Pyogenes and their by product . 1. Streptococcal sore throat :- it is the most common infection due to β-hemolytic streptococcal Pyogenes adhere to the pharyngeal epithelium by means of lipoteichoic acid-covered surface pili and also by means of hyaluronic acid in encapsulated strains . S. Pyogenes infection of the upper respiratory tract doesn’t usually involve the lungs , pneumonia when occur , is rapidly progressive and severe and is most commonly a sequela to viral infection e.g. influenza or measles , which seems to enhance susceptibility greatly . 2. Streptococcal pyoderma. - Local infection of superficial layers of skin , especially in children is called impetigo , it consist of superficial vesicles that break down and eroded areas whose denuded surface is covered with pus and later is encrusted . group A streptococcal skin infections are often attributable to M type 49,57 and 59-61 and may precede glomerulonephritis but do not often lead to rheumatic fever . C- Invasive group A streptococcal infections , streptococcal toxic shock syndrome , and scarlet fever , Fulminant invasive S.pyogenes infections with streptococcal toxic shock syndrome are characterized by shock , bacteremia respiratory failure , and multiorgan failure , death occurs in about 30% of patients , the infections tend to follow minor trauma in otherwise healthy persons with several presentations of soft tissue infection , such as necrotizing fasciitis , myositis , bacteremia occurs frequently . Pyrogenic exotoxins A-C also cause scarlet fever in association with S Pyogenes pharyngitis or with skin or soft tissue infection. The pharyngitis may be severe , the rash appears on the trunk after 24 hour of illness and spreads to involve the extremities . D- Post streptococcal diseases (Rheumatic fever , Glomerulinephritis) . following an acute S. Pyogenes infection , there is a latent period of 1-4 weeks , after which nephritis or rheumatic fever occasionally develops . the latent period suggest that the post streptococcal diseases are not attributable to the direct effect of disseminated bacteria but represent instead a hypersensitivity response . Nephritis is more commonly preceded by infection of the skin , rheumatic fever is more commonly preceded by infection of the respiratory tract . Rheumatic fever is the most serious sequel of S. Pyogenes because of results in damage to heart muscle and valves certain strains of group A streptococci contain cell membranes antigens that cross – react with human heart tissue antigens . *Streptococcal pneumonia *General characteristics : 1- It is gram-positive diplococci , often lancet – shaped arranged in chains . 2-It possess a capsule of polysaccharide that permits typing with specific antisera . 3- Pneumococci are readily lysed by surface – active agents , which probably remove or inactivate the inhibitors of cell wall autolysins . 4- Pneumococci are normal inhabitants of the upper respiratory tract of 15-40 % of human . 5- It can cause pneumonia , sinusitis , bronchitis , bacteremia meningitis and other infections processes . *morphology and identification . A- Typical organisms . - It is gram positive , lancet shaped diplococcic are often seen in young culture . - In sputum or pus single cocci or chains are also seen. - Autolysis of pneumococci is greatly enhanced by surface – active agents . - Lysis occurs in a few minutes when ox bile (10%) or sodium deoxycholate (2%) is added to a broth culture , while viridians streptococci don’t lyse and are thus easily differentiated from pneumococci . - Growth of pneumococci is inhibited around a disk of opto chin . B- Culture . - Pneumococci form small round colonies , at first dome – shaped and later developing a central plateau with an elevated rim . - It is α-hemolytic on blood agar . Growth is enhanced by 5-10 % , CO2 C- Growth characteristics . - Most energy is obtained from fermentation of glucose . this is accompanied by the rapid production of lactic acid , which limit growth . - Neutralization of broth cultures with alkali at intervals results in massive growth . D-Variation : - Pneumococcal isolates that produce large anoints of capsules produce large mucoid colonies . - Capsule production is not essential for growth an agar medium so it lost after a small number of subculture . *Antigenic structure : A- component structures : - The pneumococcal cell wall has peptidoglycan and teichoic acid , like other streptococci . - The capsular polysaccharide is covalently bound to the peptidoglycan and the cell wall polysaccharide . - The capsular polysaccharide is immunologically distinct for each of the more than 90 types . B-Quelling Reaction : - When pneumococci of certain type are mixed with specific anti polysaccharide serum of the same type or with polyvalent antiserum on a microscope slide , the capsule results markedly and the organisms agglutinate by cross-linking of the anti-bodies . - This reaction is useful for rapid identification and for typing of the organism , either in sputum or in cultures . - The polyvalent antiserum , which contains anti-body to all of the types is a good reagent for rapid microscopic determination of whether or not pneumococci are present in fresh sputum . *pathogenesis : - - - A- Types of pneumococci : In adults , types 1-8 are responsible for about 75% of cases of pneumococcal pneumonia and for more than more than half of all fatalities in pneumococcal bacteremia . In children , types 6 , 14 , 19 and 23 are frequent causes . B- production of disease : Pneumococci produce disease through their ability to multiply in the tissues , they produce no toxins of significance , The virulence of the organism is a function of its capsule , which prevents or delays ingestion by phagocytosis . C- Loss of natural resistance : - Since 40-70 % of humans are at some time carriers of virulent pneumococci , the normal respiratory mucosa may possess great natural resistant to the pneumococci . - The predisposing factor for infection with pneumococci are the following . 1- Viral and other respiratory tract infections that damage surface cells , abnormal accumulations of mucous product pneumococci from phagocytosis . 2- Alcohol or drug intoxication , which depresses phagocytosis depresses the cough reflex and facilitates of foreign material . 3- Abnormal circulatory dynamics e.g. heart failure . 4- Other mechanism e.g. malnutrition , general debility , stickle cell anemia ,hyposplenism , nephrosis or complement deficiency . *Diseases they produce : 1- pneumonia . 2- sinusitis. 3- otitis media . 4- meningitis 5- Bacteremia . 6- Septic arthritis 7- endocarditis . - The types of meningitis that caused by Streptococcal pneumonias called pneumococcal meningitis to differentiate it from meningococcal meningitis that are caused by Neisseria meningitidis . *Enterococci* *General characteristics . 1- Gram positive cocci . 2- They have the group D. group – specific substance and were previously classified as group D streptococci . 3- The structure of group D cell wall specific antigen is a teichoic acid , and this substance is not an antigenically good marker , enterococci are usually identified by characteristics other than immunologic reaction with group specific antisera . 4-They are usually part of the normal enteric flora . 5- They are usually non hemolytic , but occasionally αhemolytic . 6- Enterococci are PYR + ve . 7- They group in the presence of bile and hydrolyze esculin (bile esculin – positive ) . 8- They group in 6.5 % Nacl . 9- They grow well at between 10C0 and 45C0 whereas streptococci group at a much narrower temperature range . 10- They are more resistance to penicillin G then the streptococci , and rare isolates have plasmids that encode for β-lactamase . 11- Many isolates are vancomycin – resistant . 12- They are at least 12 species of enterococci , Enterococcus foecalis is the most common species and cause 85 – 90 % of enterococcal infections , while Enterococcus faecium cause 5-10% . 13- They are among the most frequent causes of nosocomial infections , particularly in intensive care unit . 14- Enterococci are transmitted from one patient to another primarily on the hands of hospital personnel , Enterococci , occasionally are transmitted on medical devices . 15- In patients , the most common sites of infection are the urinary tract , wounds, biliary tract , and blood . 16- Enterococci may cause meningitis and bacteremia in neonates , in adults it may cause endocarditis . 17- A major problem with the enterococci is that they can be very resistant to antibiotics . 18- E. faecium is usually much more resistant to antibiotic than E. foecalis . 19- Enterococci are intrinsically resistant to cephalosporin , penicillin , and monobactam , they have intrinsic low – level resistant to many aminoglycosides one of intermediate susceptibility to fluoroquinolones and are less susceptible than streptococci to penicillin and ampicillin . 20- The glycolpeptide vancomycin is the primary drug to a penicillin aminoglycoside for treating enterococcal infections and vancomycin resistance phenotypes are increased in frequency . 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