1MICROBIOLOGY ILaurie Baeker HovdeB.S., MT (ASCP)2GRAM STAIN• Gram stain result provides the first cluequick, easy, diagnostic toolcan guide empiric therapydivides bacteria into two groupspositive = blue or purplenegative = red or pinkdetermines cell morphologyi.e. shape, size, and arrangement3OXYGEN REQUIREMENTS• anaerobic = intolerant of O2, require proper handling and special conditions for growth• specific request required for I.D.• aerobic = utilizes O2 and grows well in an atmosphere of room air • facultatively anaerobic = will grow aerobically or anaerobically and includes most clinically significant “aerobes”4Gram positive6Gram positive•cocci (round or spherical shape)• anaerobic• Peptostreptococcus speciespart of indigenous or normal flora“opportunistic pathogens” = will only cause infection if the integrity or immunity of the host is compromised7Gram positive• cocci• aerobic• catalase test (H2O2 > H2 + O2)• positive = Staphylococcusseen as groups or clusters on Gramstained smearnormal flora of surface epithelium8Gram positive• coagulase test (an enzyme that binds fibrinogen)• positive = S. aureusa virulent pathogen• skin infections, pneumonia, osteomyelitis,food poisoning, toxic shock syndrome30% nationwide are “MRSA” • negative = “SCN”• S. epidermidis - SBE• S. saprophyticus - UTI9Gram positive• cocci• aerobic• catalase test (H2O2 > H2 + O2)• negative = Streptococcusseen scattered, in pairs, and as chains on Gram stained smearhemolytic properties on blood agar• beta, alpha, gamma10Gram positive11Gram positive12Gram positive• cocci• aerobic• gamma or alpha hemolytic• bile-esculin test (esculin hydrolysis in the presence of bile) • positive = Strep, group D13Gram positiveStrep, group D•growth in 6.5 % NaCl•PYR test (to see if the organism has the enzyme that hydrolyzes the PYR substrate)•positive = EnterococcusE. faecalis (80-90%), E. faeciumUTIs, wound infections, intraabdominal abcesses, nosocomial infections“VRE”14Gram positiveStrep, group D•growth in 6.5 % NaCl•PYR test• negative = Strep, group D,notEnterococcus (S. bovis)15Gram positive• cocci• aerobic• beta hemolytic• PYR test• positive = Strep, Group A (S. pyogenes)pharyngitis, scarlet fever, skin and soft tissue infections (“the flesh eating strep”)autoimmune sequelae = glomerulonephritis, rheumatic fever16Gram positive• PYR test• negative = Strep, group B, C, F, G, S. milleri• CAMP test (synergism between Group B Strepand S. aureus produces an enhanced arrow-shaped zone of hemolysis at the intersection)• Hippurate hydrolysis (a positive test results in a colored end product after addition of ninhydrin reagent)17Gram positive• CAMP test• Hippurate hydrolysis• positive = Strep, Group B (S. agalactiae)common (∼33%) cause of neonatal sepsis and meningitispostpartum fever and sepsisjoint infection, osteomyelitis, UTI, wound infection18Gram positive• CAMP test and Hippurate hydrolysis• negative = Strep, Group C, F, G, S. milleri• Commercial latex agglutination tests (classifies beta-hemolytic Streptococcus into Lancefield groups in about an hour)S. milleri and Strep, Group F: urogenital tract infections, abscessesStrep, Group C an G: pharyngitis, postpartum sepsis, rash, bacteremia19Gram positive• cocci• aerobic• alpha hemolytic• Optochin test (disk susceptibility test)• Bile solubility test (positive colonies autocatalyze or dissolve in the presence of bile salts)20Gram positive• Optochin test and Bile solubility test• sensitive/soluble = S. pneumoniae (pneumococci)seen as lancet-shaped pairs on Gram stained smearscommunity-acquired pneumonia,otitis media, sinusitis, meningitis∼45 % are Penicillin I or R in U.S.21Gram positive• Optochin test and Bile solubility test• resistant/insoluble = S. viridansa miscellaneous group of Strep that are part of the normal flora of oral, respiratory, and GI mucosaopportunistic pathogen, low virulencemajor etiological agent of endocarditis in the U.S.22Gram positive• bacilli (rectangular shape)• anaerobic• Clostridium speciesspore-formersfound in soil, water, dust, sewage, and in the intestinal tracts of animals and humansproduce nasty toxins that are often responsible for the symptoms23Gram positive• C. difficile• antibiotic associated diarrhea and pseudomembraneous colitis; most common agents are beta-lactams and clindamycin• nosocomial infection• carried asymptomatically as part of GI flora in up to 50% of kids < 1 year oldtissue culture = gold standard; also EIA, latex agglutination, bacterial culture for ID24Gram positive• C. perfringens• gas gangrene• third most common cause of food poisoning in the U.S. (behind Salmonella and S. aureus) characteristic double zone of hemolysisaround colonies25Gram positive• C. tetani• tetanusoften associated with puncture woundsautolysis, neurotoxin release, binding to cells in CNS, blockage of inhibitory impulses = prolonged muscle spasms• “T” in DPT vaccine26Gram positive• C. botulinum• botulismrare, but often fatalthe mechanism of action of the toxin is similar to C. tetani, binding site differs the three manifestations of the disease are food, wound, and infant botulismtrademark is acute flaccid paralysis27Gram positive• Propionibacterium, Eubacterium, Bifidobacterium, Actinomyces• normal flora, opportunistic pathogens• Lactobacillus• prevents yeast infection by competing for nutrients• found in yogurt and other foods• Mobiluncus• not part of normal flora• associated with bacterial vaginosis28Gram positive• bacilli• aerobic• Listeria monocytogenesprimary habitat is soil and decaying vegetable matterwill multiply at refrigerator temperaturescontaminated foods are the primary vehicles of transmissionlisteriosis29Gram positive• Listeria monocytogenes• seen almost exclusively in neonates, pregnant women and immunocompromised individuals• untreated/transplacental infection can lead to premature labor, septic abortion, neonatal meningitiscolony morphology closely resembles Strep, group B - must differentiate30Gram positive• bacilli• aerobic• Erysipelothrix - veterinary pathogen, human infection subsequent to animal exposure is rare• Corynebacterium • C. diphtheriae diphtheria• “D” in DPT