Microbiology: Staphylococci PDF

MICROBIOLOGY: STAPHYLOCOCCI JANREY S. DELA MERCED, RMT DIFFERENTIAL STAINING Gram staining Principal stain used for the microscopic examination of bacteria Based on the differences in composition between gram-positive cell which contain thick peptidoglycan with numerous teichoic acid cros...

MICROBIOLOGY: STAPHYLOCOCCI JANREY S. DELA MERCED, RMT DIFFERENTIAL STAINING Gram staining Principal stain used for the microscopic examination of bacteria Based on the differences in composition between gram-positive cell which contain thick peptidoglycan with numerous teichoic acid cross-linkages and gram- negative cell walls which consist of a thinner layer of peptidoglycan and lipopolysaccharides on its outer membrane GRAM STAINING Acid Fast Staining Mycobacteria and Nocardia For detection of: Acid fast Bacteria The lipid layer (mycolic acid) of the acid-fast organism takes up carbolfuchsin and resists decolorization with a dilute acid rinse. The lipid layer of the mycobacteria is of such high MW that it is waxy at room temperature and successful penetration by the aqueous based staining solutions (such as Gram's) is prevented ZIEHL- NEELSEN TECHNIQUE Micrococcae General Characteristics Catalase-positive, gram positive cocci - Spherical, pairs, Resident flora of the skin and mucous membranes Ubiquitous Infections are directly or indirectly transmitted  Staphylococcus aureus o The most significantly species, and also an important cause of nosocomial infections o Gram positive cocci arranged in tetrads or clusters and facultative anaerobic o Medium sized, raised creamy colonies on BAP or CNA with white, cream or golden yellow pigmentation; exhibits beta hemolysis on BAP  Micrococcus o Catalase positive o Coagulase negative o Gram positive cocci o Can produce yellow pigment Differential Test for Micrococcus and Staphylococcus Bacitracin Susceptibility  Peptone: 0.2% (end product: ammonia) Asaccharolytic - Does not utilize CHO as energy source Oxidizer o Organisms that utilize CHO as source of energy in the presence of oxygen Fermenter o Organisms that utilize CHO as a source of energy without the presence of oxygen Oxidizer and ferment o Utilize CHO in the presence or absence of oxygen  TEST STAPHYLOCOCCUS MICROCOCCUS AEROBIC GROWTH Facultative Strictly aerobic LYSOSTAPHIN Susceptible Resistant BACITRACIN Resistant Susceptible MODIFIED OXIDASE Negative Positive GLUCOSE Fermentative Oxidase UTILIZATION VIRULENCE FACTORS  Staphylococcus epidermidis Resembles S. aureus in morphology and gram stain Preparation Non-hemolytic in BAP, gray to white colonies Grows, but lack of fermentation in MSA Coagulase negative Susceptible to: Novobiocin Has been found with increasing frequency in immunosuppressed patients (nosocomial) Associated with bacterial endocarditis, frequently following the insertion of artificial heart valves  Staphylococcus saprophyfiticus Important cause of UTI's associated pyelonephritis and cystitis in young women and on older men who have an indwelling catheters ▪ Coagulase negative ▪ non-hemolytic ▪ Resistant to: novobiocin ▪ Slightly large colonies, with yellow pigment  Staphylococcus lugdunensis Community and hospital acquired mecA gene Causes infective endocarditis, septicemia, meningitis, UTI, septic shock STREPTOCOCCACEAE JANREY S. DELA MERCED RMT General characteristics Streptococcus and Enterococcus spp. belong to the family Streptococcaceae. Members of both genera are catalase-negative, gram- positive cocci that are usually arranged in pairs or chains. Weak false-positive catalase reactions can be seen when growth is taken from media containing blood, owing to the peroxidase activity of hemoglobin.  The streptococcal cells are more likely to appear in chains when grown in broth cultures. Most members of the genera Streptococcus and Enterococcus behave like facultative anaerobes. Some species are capnophilic, requiring increased concentration of carbon dioxide (CO2), whereas the growth of other species is stimulated by increased CO2, but CO2 is not required. CLASSIFICATION OF STREPTOCOCCI Bergey’s classificiation Lancefield classification Brown and Smith Classification Bergey’s Academic Classification Based on the physiologic divisions of Streptococcus on thermal requirement Lancefield’s Classification Based on the presence of serologically active C- CHO polysaccharide; a group of specific CHO antigens. Classified according to Rebecca Lancefield’s work (1933) Group A Rhamnose-N- acetylglucosamine Group B Rhamnose-glucosamine polysaccharide Group C Rhamnose-N- acetylglucosamine Group D Glycerol techoic acid containing alanice and glucose Group F Glucopyrasonyl-N- acetylgalactosamine Smith and Brown Classification Based on hemolytic reaction of Streptococci on Blood Agar Plate Streptococcus pyogenes S. pyogenes has a cell wall structure similar to that of other streptococci and gram- positive bacteria. The group antigen is unique, placing the organism in Lancefield group A Streptococcus pyogenes 1. M protein, encoded by the genes emm. Resist phagocytosis (resistance to infection) and plays a role in adherence of the bacterial cell to mucosal cells. More than 80 different serotypes of M protein exist. Streptococcus pyogenes 2. Adhesion molecules that mediate attachment to host epithelial cells. 3. Hyaluronic Acid Capsule. The capsule prevents opsonized phagocytosis by neutrophils or macrophages Streptococcus pyogenes 4. Streptolysin O is responsible for hemolysis on SBA plates incubated anaerobically. - refers to the hemolysin to be oxygen labile Streptococcus pyogenes 5. Streptolysin S is oxygen stable, lyses leukocytes, and is nonimmunogenic. The hemolysis seen around colonies that have been incubated aerobically is due to streptolysin S. Clinical Infections Some strains of S. pyogenes cause a red spreading rash, referred to as scarlet fever, caused by streptococcal pyrogenic exotoxins, formerly called erythrogenic toxins Appears within 1-2 days after infection Clinical Infections Bacterial Pharyngitis. The most common clinical manifestations of GAS infection Most cases of bacterial pharyngitis are due to S. pyogenes. “Strep throat” is most often seen in children between 5 and 15 years of age. After an incubation period of 1 to 4 days. Clinical Infections Clinical Infections Pyodermal Infections. Skin or pyodermal infections with GAS result in impetigo, cellulitis, erysipelas, wound infection, or arthritis.  Clinical Infections Necrotizing Fasciitis. an invasive infection characterized by rapidly progressing inflammation and necrosis of the skin, subcutaneous fat, and fascia. Many different bacteria can cause destruction of the soft tissue in this manner, a clinical feature that has been described as “flesh-eating disease.”  Clinical Infections Streptococcal Toxic Shock Syndrome. Streptococcal TSS is a condition in which the entire organ system collapses, leading to death. GAS associated with streptococcal TSS produce a streptococcal pyrogenic exotoxin, notably SpeA.  Clinical Infections Poststreptococcal Sequelae. Two serious complications, or sequelae, of GAS disease are (1) rheumatic fever and (2) acute glomerulonephritis. 1. Rheumatic fever typically follows S. pyogenes pharyngitis. It is characterized by fever and inflammation of the heart, joints, blood vessels, and subcutaneous tissues. Group A Streptococci: Streptococcus pyogenes Clinical Infections Theories: 1. Antigenic cross-reactivity between streptococcal antigens and heart tissue 2. Direct toxicity resulting from bacterial exotoxins, and actual invasion of the heart tissues by the organism. Group A Streptococci: Streptococcus pyogenes Clinical Infections 2. Acute glomerulonephritis The pathogenesis appears to be immunologically mediated. Circulating immune complexes are found in the serum of patients with acute glomerulonephritis, and it is postulated that these antigen-antibody complexes deposit in the glomeruli. Group A Streptocci: Streptococcus pyogenes SPECIMEN COLLECTION – SITE CONSIDERATIONS: OROPHARYNGEAL SWAB If the swab remains moist, no further precautions need to be taken for specimen that are cultured within 4 hours of collection. Swabs for detection of Streptococcus pyogenes are the only EXCEPTION. This organism is highly resistant to desiccation and remains viable on a dry swab for 48 to 72 hours.  Group A Streptocci: Streptococcus pyogenes Laboratory Diagnosis Cultural Characteristics. colonies are transparent to translucent, convex or domed entire, circular, shiny and surrounded by a wide zone of β-hemolysis. Group A Streptocci: Streptococcus pyogenes Laboratory Diagnosis Bacitracin (Taxo A). a presumptive test which differentiates group A from other β-hemolytic Streptococci. Principle. based on the selective inhibition of the growth of Group A streptococci by a paper disc containing 0.02 – 0.04 units of Bacitracin Results. any zone of inhibition regardless of the diameter is a (+) reaction  Group A Streptocci: Streptococcus pyogenes Laboratory Diagnosis L-pyrrolidonyl-beta-napthylamide (PYR) L-pyroglutamyl aminopeptidase BACTERIA beta-naphthylamine + cinnamaldehyde reagent (PYR reagent) PYR test. Detect the organisms ability to hydrolyze the substrate L- pyrrolidonyl-beta-napthylamide BRIGHT RED END PRODUCT Group A Streptocci: Streptococcus pyogenes Laboratory Diagnosis DICK’S TEST Test Arm Control Arm With 0.1 ml Dick’s toxin with 0.1 ml Dick’s toxoid Positive Negative Read reaction after 24 hours (+) reaction : area of irritation or redness Interpretation : susceptible to scarlet fever Group A Streptocci: Streptococcus pyogenes Laboratory Diagnosis Test to Diagnose Scarlet Fever Schultz-Charlton Reaction (Blanching Phenomenon) – based on the neutralization of erythrogenic toxins when an antitoxin is injected into the skin of a patient with scarlet fever skin rashes fade or blanch (+) – used to diagnose whether the skin rashes are due to scarlet fever or not Group A Streptocci: Streptococcus pyogenes Laboratory Diagnosis TREATMENT: – Intramuscular benzathine penicillin as single dose – Oral penicillin V for 10 days – For penicillin-allergic patients – erythromycin, clindamycin and cephalexin Group B Streptococci: Streptococcus agalactiae Group B Streptococci: Streptococcus agalactiae Virulence factors 1. Sialic acid capsule. The capsule prevents phagocytosis. Loss of sialic acid = loss of virulence 2. hemolysin, CAMP factor, neuraminidase, DNase, hyaluronidase, and protease. No evidence exists that any of these products plays a role in the virulence of this organism Group B Streptococci: Streptococcus agalactiae Virulence factors CAMP Factor (protein B) – a pore-forming protein secreted by Streptococcus agalactiae Neuraminidase – contribute to the invasiveness, cleaves the terminal N-acetylneuraminic acid Proteases – facilitate colonization on mucosal surfaces by degrading S-IgA Group B Streptococci: Streptococcus agalactiae Disease association GBS are the leading cause of death in infants in the United States. Clinical cases in newborns and is caused by vertical transmission of the organism from the mother. Colonization of the vagina and rectal area with GBS is found in 10% to 30% of pregnant women.  Group B Streptococci: Streptococcus agalactiae Laboratory Diagnosis Culture Media Todd-Hewitt broth – 10 ug/ml colistin and 15 ug/ml nalidixc acid – Alternative: Gentamicin 8 ug/ml – substitute for colistin StrepB Carrot Broth (SCB) – Produced orange or red pigment even after 6 hours of incubation Group B Streptococci: Streptococcus agalactiae Laboratory Diagnosis Cultural characteristics The GBS grow on SBA as grayish white mucoid colonies surrounded by a small zone of β- hemolysis. Group B Streptococci: Streptococcus agalactiae Laboratory Diagnosis Presumptive identification is based on biochemical reactions. The most useful tests are positive hippurate hydrolysis and CAMP tests. These tests enable the organism to be readily differentiated from other β-hemolytic streptococcal isolates. Group B Streptococci: Streptococcus agalactiae Laboratory Diagnosis CAMP (Christie, Atkins, and Munch-Peterson) test – Principle: The CAMP factor is a diffusible, protein-like compound produced by Streptococcus agalactiae. A characteristic “arrowhead” hemolytic pattern results when the organism is streaked perpendicularly to β- hemolytic S.aureus. Group B Streptococci: Streptococcus agalactiae Laboratory Diagnosis IDENTIFICATION Hippurate Hydrolysis Principle: 1% Sodium hippurate hippurate hydrolase BACTERIA Glycine and Benzoic acid  Group B Streptococci: Streptococcus agalactiae Laboratory Diagnosis IDENTIFICATION Hippurate Hydrolysis Principle: 1% Sodium hippurate hippurate hydrolase → BACTERIA Ninhydrin purple colored product Glycine and Benzoic acid (37°C for 2 hours) (37°C for 10 minutes) Group B Streptococci: Streptococcus agalactiae Treatment The clinical response to antimicrobial therapy is often poor despite heavy doses given. Some clinicians recommend a combination of ampicillin and an aminoglycoside for treating GBS infections. Alpha-hemolytic: Streptococcus viridans Streptococcus pneumoniae Alpha-hemolytic: Streptococcus viridans The term viridans means “green,” referring to the α-hemolysis many species exhibit. GROUP: (1) S. mitis group (including S. mitis, S. pneumoniae, S. sanguis, S. oralis); (2) S. mutans group (3) S. salivarius group (4) S. bovis group (5) S. anginosus group  Alpha-hemolytic: Streptococcus viridans Disease association 3 Main Infections Caused by Viridans Streptococci Dental Infections – Bind to the teeth and ferment sugars, which produces acid and dental caries (cavities) Endocarditis – Dental manipulation sends showers of these organisms in the blood stream. Abscesses – Most frequently caused by Streptococcus intermedius group (S. intermedius, S. constellatus, and S. anginosus) which are microaerophilic.  Alpha-hemolytic: Streptococcus viridans Laboratory Diagnosis It is extremely difficult to identify isolates of the viridans group to the species level; clinical laboratories should be satisfied to place isolates into one of the five groups. All members are PYR negative and leucine aminopeptidase (LAP)- positive. Alpha-hemolytic: Streptococcus pneumoniae Alpha-hemolytic: Streptococcus pneumoniae Also known as pneumococcus, S. pneumoniae is isolated from a variety of infections. S. pneumoniae is a member of the S. mitis group, but it is much more virulent than other members of the group. S. pneumoniae can express one of approximately 90 different capsular types. Antibody directed against the capsular antigen is protective. Alpha-hemolytic: Streptococcus pneumoniae Determinants of Pathogenicity PATHOGENESIS: Invasion of the Host Tissue (1) Neuraminidase – contribute to the invasiveness, cleaves the terminal N-acetylneuraminic acid (2) Autolysin – release pneumolysin O (3) H2O2 – damage to host cells (4) Pneumonysin O – lysis of the host cells Alpha-hemolytic: Streptococcus pneumoniae Determinants of Pathogenicity As the colonies become older, autolytic changes result in a collapse of the center of the colony (with raised margins and depressed centers), giving it an umbilicate or doughnut appearance (checker or nailhead colonies). Alpha-hemolytic: Streptococcus pneumoniae Determinants of Pathogenicity PATHOGENESIS: Attachment to the Host Tissue (1) Proteases – facilitate colonization on mucosal surfaces by degrading S-IgA (2) Pili – contribute to colonization and increase the formation of large amounts of TNF by the Immune system.  Alpha-hemolytic: Streptococcus pneumoniae Determinants of Pathogenicity C-substance – a component of the cell wall of pneumococci which is a teichoic acid that reacts with some components of the immune response resulting in the activation of some nonspecific host immune responses Similar to the C-CHO of other streptococci Alpha-hemolytic: Streptococcus pneumoniae Disease Association – Most common cause of bacterial pneumonia (Lobar) – Second most common cause of bacterial meningitis – Otitis media, purulent sinusitis and occasionally peritonitis Alpha-hemolytic: Streptococcus pneumoniae Laboratory Diagnosis Specimens: sputum, swabs, pus and blood Gram-stained smears Culture Alpha-hemolytic: Streptococcus pneumoniae Laboratory Diagnosis Optochin Test – Most widely used presumptive test for differentiating S.pneumoniae from other alpha- hemolytic streptococci (e.g., the viridans streptococci) Contains ethylhydrocupreine hydrochloride A.k.a = Taxo P Alpha-hemolytic: Streptococcus pneumoniae Laboratory Diagnosis Positive reaction is a 14-16 mm zone of inhibition using a 6mm Optochin disk. Negative: No zone of inhibition Equivocal: Any zone of inhibition less than 14 mm is questionable for pneumococci; the strain is identified as a pneumococcus only if it is bile- soluble.  Alpha-hemolytic: Streptococcus pneumoniae Laboratory Diagnosis Bile Solubility Test Principle S. pneumoniae produce a self-lysing enzyme to inhibit the growth The presence of bile salt accelerate this process Procedure: Add ten parts (10 ml) of the broth culture of the organism to be tested to one part (1 ml) of 2% Na deoxycholate (bile) into the test tube Negative control is made by adding saline instead of bile to the culture Incubate at 37oC for 15 min and Record the result after 15 min  Alpha-hemolytic: Streptococcus pneumoniae Laboratory Diagnosis Results: – Positive test appears as clearing in the presence of bile while negative test appears as turbid – S. pneumoniae soluble in bile whereas S. viridans insoluble  Alpha-hemolytic: Streptococcus pneumoniae Laboratory Diagnosis Inulin Fermentation Test Principle: – Based on the ability of the organism to ferment Inulin, a naturally occuring polysaccharide of plants – Results: Change of color of pH indicator and bubble/gas formation which indicates that the sugar is fermented and acid production results.  Alpha-hemolytic: Streptococcus pneumoniae Laboratory Diagnosis Neufeld Quellung (Capsular Swelling) – Principle: A biochemical reaction in which anticapsular antibodies bind to the capsule of a bacterium, resulting in the capsule to swell or become more visible, especially under the microscope. Reagents: –Anti-sera containing anticapsular antibodies –Methylene blue.  TESTS TO DIFFERENTIATE PNEUMOCOCCI FROM STREPTOCOCCI MIBON-Q Tests Pneumococci Streptococci Mouse Virulence Test Mouse dies within 16-48 hours won’t die Inulin Fermentation Test fermenter non-fermenter Bile solubility Test bile soluble insoluble Optochin Test susceptible resistant Neufield-Quellang Capsular Swelling Test swelling of capsule no swelling STREPTOC OCCACEAE Gamma-hemolytic: Enterococci Gamma-hemolytic: Enterococci Enterococci possess the group D antigen. S. bovis is no longer a valid species name. Group D streptococci were subdivided into the enterococcal and nonenterococcal groups.  Gamma-hemolytic: Enterococci Disease Association Normal flora of the skin, upper respiratory, gastrointestinal and genitourinary tracts Diseases produced: frequent cause of bacterial endocarditis in the elderly urinary and biliary tract infections septicemia wound infection and intra-abdominal abscesses  Gamma-hemolytic: Enterococci Treatment Enterococcal strains generally resistant to penicillin G, ampicillin and penicillinase-resistant penicillins penicillin in combination with an aminoglycoside (gentamicin or streptomycin) for penicillin-allergic patients – vancomycin and erythromycin Nonenterococcal strains – penicillin G Gamma-hemolytic: Enterococci Laboratory Diagnosis Both groups were bile esculin- positive, but the nonenterococcal organisms would not grow in a nutrient broth with 6.5% NaCl.  Gamma-hemolytic: Enterococci Laboratory Diagnosis Bile Esculin Growth w/ 6.5% NaCl PYR Enterococci + + + Non-Enterococci + _ _