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Neisseria, Haemophilus and Bordetella World of gram- negatives!!  Neisseria General characteristics: - aerobe? Fac anaerobe? Anaerobe? - gram-negative cocci (0.6 -1 µm in size) - arrangement: in pairs with flattened sides adjacent to each...

Neisseria, Haemophilus and Bordetella World of gram- negatives!!  Neisseria General characteristics: - aerobe? Fac anaerobe? Anaerobe? - gram-negative cocci (0.6 -1 µm in size) - arrangement: in pairs with flattened sides adjacent to each other - non-motile (cocci = non-motile) - catalase? - oxidase? What is this enzyme? cytochrome oxidase- electron transport chain - OXIDIZE carbohydrates (do they ferment?) ** use sugar in presence of O2 and produce acid - some: normal flora of upper resp. tract - others: OBLIGATE human pathogens! Fig. 29-1  Neisseria: physiology Two medically relevant species that are pathogens : N. meningitidis causes meningitis (nasopharynx of healthy people) can colonize WITHOUT disease N. gonorrhoeae causes gonorrhea (common STD) strict pathogen (what does this mean???) Loading… N. gonorrhoeae: fastidious, what are the growth requirements? What about CO2 need addition of cysteine fatty acids are toxic (starch added to media to neutralize fatty acids) Optimum growth temp: ? At what temperature won’t they grow? Difficult to grow easy to transmit from person to person  Neisseria: structure Typical gram-negative: thin peptidoglycan + outer membrane ** LPS of Neisseria is called lipooligosaccharide (LOS): why?? have highly branched sugar chains (oligosaccharides) ** does lipid A still have endotoxic effect?! N. meningitidis: covered in carbohydrate capsule N. gonorrhoeae: no true capsule but sugars with negative charge on surface Surface and membrane proteins: - pili: attachment, transfer of genetic material we make antibodies against pili of but why do antibodies not protect? - porin: form pores and channels in outer membrane - Rmp protein: antibodies to this interfere with other antibodies we make to cure ** thus antibodies to Rmp HELP pathogenesis!!!*  pil pil i i Loading…  Neisseria: pathogenesis I. General Traits: - Require iron: compete with humans for their iron how does this relate to their ability to only infect humans? - Produce IgA protease: inactivates IgA - Surface proteins mediate attachment and penetration of host cells Meningococcal pili: preferentially bind cells in nasopharynx - N. gonorrhoeae: internalized by phagocytes and survive what happens then?? II. Immune Responses: - LOS from both pathogens stimulates inflammation - IgG: primary antibody produced against N. gonorrhoeae against: Omp’s, LOS, pili, capsule - Antibodies to Rmp block bactericidal antibody response ** Protection against meningococci: humoral or cellular?? -Meningitis: occurs when antibody response is low (i.e. children) why are some infants initially protected?  Neisseria meningitidis -a.k.a. the “meningococcus” - CAN it be carried without symptoms? - large antiphagocytic capsule (how is this different from gonococcus) - VERY sensitive to temps above or below 37C (unusual) - undergoes autolysis: release of endotoxin during meningitis and septicemia - colonizes nasopharynx: of what animals? - Causes: meningitis (inflammation of the meninges: membrane surrounding brain and spinal cord) meningococcemia: characterized by skin lesions - can survive within what type of immune cells?  Meningitis - Infection: bacteria are _______, attach to what cells?, cross mucosal barrier enter bloodstream - Meningitis: mildest form: fever and malaise: resolves in 1-2 days - Fulminant meningitis: (serious) form: fever, vomiting, headache, seizure, coma - Initial symptoms of FULMINANT meningitis: Infants: Older children: more specific symptoms severe headache, fever, ? Progression: neurological damage, convulsions, spinal rigidity hamstring spasms, can this kill you? - Meningococcemia: septicemia but no meningitis (5-15% of patients) - Hemorrhages of the skin in 30 to 60% of patients - Pulmonary insufficiency (vascular coagulation) develops how quickly? - Many die within 24 hours of the onset of these symptoms  Neisseria gonorrhoeae - The “gonococcus” does not colonize without disease - STRICT human pathogen: remind yourselves what this means? - causative agent of gonorrhea: how common is this infection? 2nd most in us commonly spread STD - transmitted primarily by sexual contact woman have 50% chance of contracting after single exposure men have 20% chance of contracting after single exposure -Major reservoir is: who? > - is the a sympormaic carrier - 358,000 cases reported in U.S. (www.cdc.gov): underestimates true numbers! - rates of infection highest in what part of US? - lowest numbers in what part of US?  Gonorrhea -Specific type of urethritis: involves mucous membranes of urethra **results in large volume of pus discharge from urethra** -Infects superficial columnar epithelial cells: urethra, cervix, rectum, pharynx and conjunctiva - Men: - 20% chance after 1 exposure (80% with > 4) - infection limited to what body part? pus WBC dead - purulent discharge: how many days after infection?? = Inflammation , , Loading… - what % of infected men display acute symptoms? - Women: - what body site is infected? endocervical infection - purulent discharge, abdomnial pain and dysuria (painful urination) - how many women are asymptomatic? 50 % of women a symptomatic - untreated: pelvic inflammatory disease, ovarian abcess: 10-20% of women fallopian tube inflammation, endometriosis Get gonorrhea from Mother V Ocular infections: newborns commonly exposed in birth canal - causes purulent conjunctivits F eyes - Group B Streptococci + gonorrhes Pharyngeal infections Midterm ; What other bacteria tested are women  Laboratory Diagnosis Gonorrhea: SAMPLE what body sites? = male urethra sample and female endocervic Use Gram stain: Sample Male symptomatic urethritis: > 90% specific and sensitive All negative Male ASYMPTOMATIC urethritis: < 60% sensitive results confirmed with Women: insensitive: collect enough specimen from cervix culture DNA probes and monoclonal antibodies Meningitis: Organism readily detect Cerebrospinal fluid: do you find them readily?# Bacteremia: are there many organisms in blood? Many organism in blood (unsua) Culture: * DO = Gram stain blood bla Many Dacteria NOT N. gonorrhoeae: readily cultured from genital specimens Selective AND non-selective media: why? too many organisms on mucosal surfaces - [Gonococci: very sensitive to drying and fatty acids Meningococcus: less sensitive to these conditions: care with virulent strains!  Laboratory Culture and Identification Gonococci die if exposed to drying or cold temperatures Gonococci blood cultures: *must be careful with blood culture media: can be toxic to bacteria** Identify PATHOGENIC Neisseria on basis of: (Table in Ch. 29) - fastidious growth: poor growth on Nutrient agar (use what media?) - gram-negative diplococci - oxidase ?, catalase ?  Treatment and Prevention vaccine Gonorrhea: * on quit cause , treatment , resistant - Many gonococci are resistant to what antibiotic? penicillin - CDC recommendation: third generation cephalosporin e.g. ceftriaxone or ciprofloxacin (“Cipro”) treated - Both partners must be treated or what is likely? Both partners must be or reinfection rate is high Meningococcal meningitis: - Susceptible to what antibiotic? - what antibiotic can cross blood-brain barrier in this case? Why **usually would not** penicillin cross blood-brain barrier can when Meninges is inflamed - Chloramphenicol or 3rd generation cephalosporin if allergic to ? penicillin Control: Gonorrhea: vaccine???? No vaccine yet - need education, aggressive detection and follow up screening vaccine Meningitidis: can’t control reservoir (healthy carriers) Yes , need prophylaxis for healthy people exposed to the sick Vaccine: against 5 serotypes for children > 2 yrs old Haemophilus  not cause the fill Does HaemophiluS = Overcook s blood General characteristics: Chaco - gram-negative RODS: typical G- wall and membrane - outer membrane: contains LPS (endotoxin) and strain-specific proteins - - non-spore formers - non-motile* gram negative rod but does not move cocobacilary - shot stubby rod - aerobic or facultative anaerobes * Most Niseric aerobic are - fastidious: need hemin (a.k.a. X factor) NAD (nicotinamide adenine diphosphate): a.k.a. V factor # I ove energy z In lab * or species v factor required Supplied in sheep blood: MUST BE HEATED FIRST why? remove to NAD > heated blood agar: called what? Chocolate Agar inhibitors Some have capsule: 6 different serotypes ** nonencapsulated (without capsule) still cause disease (contrast with??) major vaccines ↓ Vaccine: 1 serotype vaccine: type B = strep aniemo need capsule to cause HIB disease  Haemophilus: epidemiology Commonly colonize mucous membranes in upper respiratory tract (healthy): H. parainfluenzae and nonencapsulated H. influenzae - H. parainfluenzae: 10% of saliva flora - can spread locally: ears: otitis media sinus: sinusitis lower r.t.: bronchitis and pneumoniae - disseminated disease: RARE Contrast with ENCAPSULATED H. influenzae serotype B: - most common disease-causing serotype - is it common in upper respiratory tract? uncommon - common cause of meningitis: unvaccinated or elderly - Vaccine (1987): how old do you have to be to get the vaccine? 2 months -> of age incidence of systemic infections was 20,000/year how many after vaccine? = 30 year - Developing nations (no vaccine): 1 of the most important pediatric infections 3 million cases/year how many deaths per year??  Haemophilus: pathology upper respiratory tract Type B: adherence to URT with pili and other adhesins Cillia ↓ LPS: impairs what cell or structure? leading to epithelial cell damage also induces inflammation Translocates into blood: without opsonic antibodies - bacteremia Capsule -is this a major virulence factor? Major virulence factor contains sugar: polyribitol phosphate (PRP) antibodies to PRP: stimulate complement mediated killing stimulate phagocytosis Severity of disease correlated with rate of bacterial clearance from blood: ↑ ____ meningitis risk ↓ if ____ antibodies to PRP More antibiotic diease you have less  Haemophilus: diseases (infections) H. influenzae: Encapsulated: meningitis: primarily affects what type of children? unvaccinated children same symptoms as other types of bacterial meningitis 1-3 day initial mild symptoms -meningitis symptoms Recovery: ? with proper intervention 90% With proper intervention epiglottitis: inflammation of epiglottis (cartilage behind tongue over larynx) is this life-threatening? Yes rapid obstruction of airways , Died by suffocation no of to brain arthritis: before vaccine: most common arthritis must add supplements! Chocolate agar or Levinthal’s agar Staphylococci: lyse blood cells and release Factors V and X Factor V NAD (nicotinamide adenine dirnucleotide) if grown anaerobically: do they require X factor??? NO Themin Agglutination: antibodies on latex beads for detection of PRP (capsule antigen) ** limited: only available for H. influenzae type b**  Haemophilus: treatment and prevention Antibiotics of choice: Meningitis and epiglottitis: PROMPT treatment (mortality?) 100 % mortality without Treatment: cephalosporins (broad-spectrum) Sinusitis and otitis: treat with ampicillin (if sensitive) Chancroid: erythromycin capsule not live bacteria ↓ Prevention: vaccine against H. influenzae type b (conjugate of pure capsular PRP) dosage: 3 doses vaccine before 6 months old HID vaccine Bordetella  Bordetell a General characteristics: - gram-negative coccobacillus - strict aerobes - simple nutritional requirements *add charcoal, starch, blood to absorb any toxic compounds Most important species: B. pertussis: what’s the major disease? Whooping cough ↓ - exclusive human pathogen Bordetella - vaccine available (DTP): diphtheria-pertussis-tetanus) 1949 ** still ~60 million cases/year worldwise ** 12,000 cases/US distinguished from other Bordetella species: (Table 35-2) oxidase + urease - non-motile no growth on sheep blood agar  Bordetella pertussis: wooping cough - Bacteria inhaled into R.T. - Binds to ciliated cells lining airways - Damages cells with the following toxins:. toxin 3 1. Pertussis toxin: two component toxin enters cells and increases cAMP - increases respiratory secretions/mucus 2. Tracheal toxin: directly interferes with DNA of ciliated cells cilia? inhibits cilia Movement and eventually destroys ciliated cells interference with clearing mechanisms - leads to ?? "Whooping cough 3. Hemagglutanin: promotes binding to membranes of ciliated cells Increase Mucus  Whooping cough: disease progression Need to know the different 7-10 day incubation after inhalation three cyle of whoop Followed by three stages of disease: Catarrhal: I 1 peak # of bacteria disease may not yet be diagnosed infectivity?? - high risk Paroxysmal: coughing attacks - mucus clearance impaired **classic whooping cough -vomiting (?) and exhaustion - 40-50 paroxysms/day peak Convalescent: coughing stop - paroxysms subsiding lag - secondary complications only happen when treatment not Not diagnosis b/c of the whooping sound Fig 35-2 seen  Specimen collection and diagnosis Specimens: -extremely sensitive to what condition?? to drying - optimal specimen: ? nasopharyngeal ** no cotton swabs: contain fatty acids that are toxic to bacteria calcium alginate or fiber swabs needed - Plate immediately (AT BEDSIDE) or into specialized transport media - chocolate HORSE blood agar or Regan-Lowe charcoal Microscopy: use fluorescent antibodies on direct smear of sample Culture: chocolate HORSE blood agar or Regan-Lowe charcoal long incubation (~ 7days) humid incubation conditions Nucleic Acid amplification: PCR or other methods to specifically detect B.p. DNA ** recommended diagnostic!!**  Whooping Cough Treatment - Supportive therapy Stage Early - Antibiotics could help if given early: most not diagnosed at catarrhal stage macrolides are the choice: erythromycin, azithromycin (“Z-pack”) - Recovery depends on regeneration of what type of cells? & ciliated cells - Vaccine: inactivated pertussis toxin + bacterial component 1. DTaP 5 doses: 2, 4, 6, 15 months and one between 4 and 6 years 2. Tdap: booster for adolescents (11-12 yrs) and pregnant women Recent outbreaks: 48,000 cases of whooping cough reported in 2012! (up from 18,000 cases reported in 2011!!) Why? increased awareness, improved diagnostic tests, better reporting, more circulation of the bacteria, and waning immunity from vaccines infants and very young =>highest risk of serious illness BUT! adolescent cases (13-14 yr olds) are INCREASING!  Corynebacteriu · No spores m Gramtrod - Gram-positive rods - clumps, short chains (V or Y arrangement), irregular, club shaped - Aerobic or facultative anaerobes, catalase + - Do not form spores - High G+C content in DNA - Unique cell wall: diaminopimelic acid and mycolic acid stain with methylene blue ↑ - Metachromatic granules can be seen inside some *stain differently than primary dye* ↑ up dye soak bacteria Y or rod -Normal colonizers: skin, upper RT, GI, urogenital * opportunists ↑ can become T Respirory tract diasease -Pathogen: C. diphtheriae causes diphtheria  Corynebacterium diphtheriae -Irregular-club shaped rods - Metachromatic granules visible after staining with methylene blue granules: inorganic polyphosphates (volutin) serve as energy reserves and are not membrane bound - diphtheria-tetanus - Pertussis -Pathogenesis: (DTP vaccine prevents) Two-component exotoxin - encoded by bacteriophage - exotoxin expressed when iron concentrations are LOW - => Loading… - enters target cells: stops protein synthesis in cell Phospholipase D: increases permeability of blood vessels - allows organism to spread WITHIN NASOPHARYNX Epidemiology: ↓ no symptoms ↓ snezzing - carried asymptomatically in throat spread via respiratory droplets and coughing = - sore throat, low grade fever = grey, pseudomembrane over throat bacteria, lymphocytes, plasma- cells, fibrin, and dead cells Mix Dying bacteria , WBC form membrane - toxin causes: vessel damage, bleeding, myocarditis, trouble breathing back Of - toxin can spread through body =death from toxin-mediated heart failure throat Epidemiology: - carried asymptomatically in throat spread via respiratory droplets - sore throat, low grade fever grey, pseudomembrane over throat - bacteria, lymphocytes, plasma cells, fibrin, and dead cells - toxin causes: vessel damage, bleeding, myocarditis, trouble breathing - toxin can spread through body death from toxin-mediated heart failure Grey Pseudomembrane  Diagnostics Confirmed diagnosis of diphtheria: - only by isolating toxigenic diphtheria bacilli from the primary lesion (e.g. throat) Microscopy: Methylene blue stain shows metachromatic granules Gram stain: Gram-positive pleomorphic rods arranged in irregular formations Culture: Growth media: (cysteine or serum-) tellurite agar F Differential C. diphtheriae produce gray to black colonies on the tellurite media tellurite is reduced intracellularly to tellurium Any colonies which appear: stain with toluidine blue O or methylene blue Differentiate from Arcanobacterium (box 27.1): catalase-negative no DAP in cell wall Reverse CAMP TEST Arcanbacterium : Cat neg ; no DAP in cell wall Reverse CAMP ; test. pseudotuberculosis C (box 27. 1) * Reverse Camp test * Legionella  Legionella: gets its name from 1st outbreak American legion 1976 General characteristics: Hard to find and grow! - gram-negative poorly staining pleomorphic (coccobacilli) rods - obligate aerobes and nutritionally fastidious -Media requires L-cysteine and iron -buffered charcoal yeast extract agar 58 species: Water, lakes cooling towers/air conditioners showers hot tubs. **Human exposure through contaminated aerosols and mists *** Not spread from person to person L. pneumophila: Legionnaires disease and Pontiac fever: 18,00 cases/year USA - Infect free living amebae in nature /biofilms - Infect alveolar macrophages, monocytes and epithelial cells Survive intracellularly by preventing lysosomal fusion Asymptomatic Infections: in healthy patients. Pontiac fever:1-2 days Influenza like: high fever, chills headache with no pneumonia Legionnaires disease: 2-10days a severe form of pneumonia leading to multiorgan infections Death 15% healthy and 75% in immunocompromised  · Bioflin in Diagnostics are present water Microscopy: Stains poorly and hard to find in tissue sections Culture is gold standard: 3-5% CO2 3-10 days Growth media: Buffered charcoal yeast extract agar Media requires L-cysteine and iron Test growth vs no growth on non supplemented media Will not grow on Sheep's blood agar Serologic testing: Legionella urinary antigen test for initial detection Nucleic acid amplification and PCR: Respiratory samples and water Antibody titers at 4x normal but not reliable Treatment Macrolides or fluoroquinolones Prevention and screening in water supply Water cooling tower testing: NYC –PHL labs Water systems monitored in hospitals and nursing homes Treatment by hyperchlorination, heating, biocides and coper-silver ionization.  Antimicrobials = a substance that Ch. 20 and Bartelt: 22 and 23 kill or inhibits the growth * Define May type of antibiotic Cell Wall Resistance  Antibiotics: general Antibiotics: aka antimicrobial , antibacterial Broad definition: compound produced by microbe to kill or inhibit Microbe bacteriacidal: Kill Organism ; Cidal> Kill bacteriostatic: inhibits growth ; static > growth - - Before 1930: antiseptics 1867 LISTER (carbolic acid) - - nothing for systemic & infection apply topically skin 1930’s: Sulfa drugs discovered by accident Fleming ↑ discover penicillin 2008: many antibiotics (specific and broad-spectrum) AND antibiotic resistance! Classify based on mode of action: Four specific areas targeted by antibiotics: - Cell wall Synthesis protein Synthesis - for quiz - nucleic acid Synthesis ask - metabolite Synthesis  Inhibitors of cell wall synthesis Examples: penicillin, cephalosporins, vancomycin, bacitracin, polymixin, isoniazid ↓ - ↑ B-lactams glycopeptide Polyperide mycolic acid inhibitor antibiotics β -lactam antibiotics: contains LACTAM ring Mycrobacice inhibits cell wall synthesis Cell wall peptidoglycan; = Reminder - NAM-NAG repeating units Loading… - Cross linking catalyzed by groups of enzymes Enzymes coordinated by: peniclin binding protein (PBP) β –lactam antibiotics: Bind to PBP: inhibit PBP cells die cell and autolusin fall apart bactericidal or bacteriostatic?? contrast to bacteriostatic  Inhibitors of cell wall synthesis (cont’d) 1. β -lactam antibiotics: inhibit cell wall synthesis: bactericidal active against what type of cells?? growing cell A. Penicillin: very effective against which organisms F gram ; low toxicty - produced by fungus: Penicillium chrysogenum - changes in β-lactam ring creates new derivatives Types (e.g.) Penicillin V: prefered form ( Ampicillin: broad-spectrum ; activity against gram Carbenicillin: more broad spectrum ; More grom heg B. Cephalosporins: - produced by fungus: Cephalosporium - mode of action? Spectrum of organisms it is active against? - 3rd and 4th generation: very broad range of gram-negatives ** many g-negatives already developing resistance**  Loading… Antibiotic Resistance to β-lactam antibiotics  Mechansims of resistance: β-lactam antibiotics Paths to resistance: 1. Hydrolyze antibiotic with enzymes: B-lactamases *group of different enzymes, specific for particular antibiotic "group e.g. Class B β-lactamase: wide range of activity against all B-lactams on plasmid transferred : easily 2. Prevent antibiotic from reaching PBP between bacteric ** resistance seen in which organisms? grant due to OM how does it penetrate and how does that relate to resistance? Must penetrate om via porins : Dacteria modify poring 3. Modify ability of antibiotic to bind to PBP a. NEW PBP = aquired can bind not antibiotics (MRSA) b. modify PBP: via recombination existing or mutation  Inhibitors of cell wall synthesis: NON β-lactam antibiotics Glycopeptides: inhibt Cell Wall Synthesis bacterial : e.g vancomycin: disrupts peptidoglycan in grown gram pos interferes with Ala-Ala in side chains * prevent cross bridge from forming properly Not effective against: - gram-negatives: to big pen OM - organisms that have different side chains (no Ala-Ala) - Certain Enterococcus strains: acquired Resistance Polypeptides: examples bacitracin: topical for G Skin infections (t-resistance) + polymixin: cyclic peptide: insert into OM and increase permeability eventual cell death * effective * against G- * * nephrotoxic  Inhibitors of Protein Synthesis 1. Aminoglycosides: Dacterial e.g. streptomycin, kanamycin, erythromycin, gentamicin, tobramycin Mechanism of action: Pass through OM, cell wall, and membrane: Oxygen dependent process Which organisms are resistant? Why? anaerobe resistant Bind what? Why does this end protein synthesis? = Irreversibly Tos subunit of ribosome protein synthesis Use: Gram-negative and SOME gram-positive: *op Enterococci : Streptococci : cell wall thick 2. Tetracyclines: REVERSIBLY bind to what? 305 subunit ribosome broad spectrum: Bacteriostatic e.g. tetracycline, doxycyline Tetracycline toxicity: not used for < 18 yrs old--- WHY NOT?  Inhibitors of Protein Synthesis (cont’d) 3. Linezolid: blocks tRNA, mRNA and ribosome complex by = bind to 50 s ribosome ** no protein synthesis: unique mode action inhibit assembly · : of ribosome active against: Enterococcus*, Staphylococcus, Streptococcus 4. Chloramphenicol: broad spectrum bactericidal - binds to 50S ribosomal subunit reversiply , - Toxic effects (not frequently used in U.S.) disrupts protein synthesis in bacteria AND humans: * bone marrow suppression: reversible ; close dependent * idiosyncratic aplastic anemia: inreversible ; does independent WBL RBC plated formation - , dispont , - usually fatal 5. Macrolides: bacteriostatic, broad spectrum (but many g-negatives now resistant) binds to 23S rRNA of 50S ribosomal subunit: REVERSIBLY e.g. erythromycin and azithromycin (Z-PAK)  Loading… Antibiotic Resistance to Protein Synthesis Inhibitors  Mechansims of resistance: & Protein synthesis inhibitors Paths to resistance: 1. Mutation of the ribosome binding site for antibiotic F Do Not say Mutate target cell 2. Limit uptake of antibiotic into bacterial cell F 60 3. Increase expulsion of antibiotic from cell Imme Most common 4. Modify antibiotic with enzyme so it can’t bind ** most common  Inhibitors of Nucleic Acid Synthesis Quinolones: most widely used class of antibiotics : bactericidal - active against? G and G + - - inhibit DNA synthesis how? = by blocking enzyme involve in > DNA replication, recombination and topoisomerase - e.g. ciprofloxacin: not used for < 18 yrs or pregnant Why not? damage the carriage of young resistance: mutate DNA synthesis enzymes decrease drug uptake or increase expulsion from cell 2. Rifampin: inhibits RNA synthesis from DNA: bactericidal ** Mycobacterium tuberculosis (TB) resistanceoccur Combination With rapidly : other antibiotic * target Cul 3. Metronidazole: used almost exclusively for parasite and Anarobic bacteria ** activity: produce toxic compounds that disrupt bacterial DNA * vaginitis, amebiasis, Giardia infections, anaerobes  Antimetabolite antibiotics Sulfonamides: prevent synthesis of folic acid needed by certain microbes *need folic acid for ?? Susuthesic amino acid and nucleic acid humans: why does antibiotic have no adverse effect on humans? don't systheis folic acid (B9) e.g. trimethoprim and dapsone no adverse effect broad spectrum: many gram-positive AND negative can be hupersensitive *short acting sulfonamides (e.g. sulfosoxazole): UTI by E Coli *Chronic or long term UTI: + rimethoprim treatment Enterococci and Pseudomonas are intrinsically resistant Antimicrobial Susceptibility Tests Bartelt (Lab manual): Ch. 23  Antimicrobial susceptibility tests (AST): The “who”, “what” and “how” Who: National Committee for Clinical Laboratory Standards (NCCLS): Provide Ast guidelines antibiotic · : type of Standardization performance and reporting , , The “what”: 1. Reporting results: of antimicrobial susceptibility test - susceptible: treat particular antimicrobial - resistant: cannot be treated particular antimicrobial - intermediate: antibiotic effective under what conditions? higher dose or concentrated 2. Test performance indications: i.e.: should an AST be performed? - what are the criteria? only if a standarized - what factors about the bacteria influence test choice? 3. Selection of antimicrobials to test: - Who determines the tests to perform? What factors are considered - Are test choices standard across all labs? Why or why not? 4. Selective reporting:  Standardization ** Strict adherence to standardized test methods: requried for Ast performan Specific procedures developed for which aspects of testing? 1. Culture media. 3 incupaction condition. 2 inoculum Standard preperation = 35 · 0 1. Culture media Mueller-Hinton agar: standard for which organisms? Which components of the media can produce unwanted/inconclusive results? ** Other specialized media for other organisms 2. Inoculum: McFarland turbidity standards: what are these? Each standard is numbered: from 0.5 and up 0.5 McFarland standard = ? “Standardized suspension”:  Standardization (cont’d) 2. Inoculum (CONT’D): Considerations: - Purity - Why are many colonies used sometimes? 1. Log phase growth method: inoculate broth with a few organisms grow to turbidity =? 2. Direct colony suspension: inoculate broth with many colonies- how is this standardized from test to test? 3. Stationary phase growth method: small broth inoculated growth before inoculating?  Antimicrobial test methods Many different types: discuss only a few 1. Broth dilution tests: what types of results are produced? - various antibiotic concentrations tested on broth cultures antibiotic concentrations: how are these expressed?? - How do you prepare dilutions of the antibiotic? - How is test performed? a. Uninoculated broth culture: volume and concentration of antibiotics? b What is inoculum what is the standardized suspension? large volumes:? ; small volumes: ? d. Incubate and read results: check purity; controls: no antibiotics, no inoculum ** Determine MIC: ? definition: ? MIC values: how are the values classified and on what is this based? How do you report the results?  Antimicrobial test methods (cont’d) Many different types: discuss only a few 1. Broth dilution tests: 2. Disk diffusion tests (“Kirby Bauer” test): what type of results are obtained? - paper disks impregnated with antibiotic: - placed on bacterial lawn: how do you prepare inoculum for the lawn? - disks firmly and evenly placed: not relocated after placed - zone of inhibition Principle: - Concentration gradient of antibiotic - Where does growth appear? Interpretation: zone size: how do you report the results?  Antimicrobial test methods (cont’d) Many different types: discuss only a few know the differierc 1. Broth dilution tests: 2. Disk diffusion tests: 3. E-tests: what type of results are provided? -how does this differ from disk diffusion? How is it similar? -how do you determine results? MIC NO MVC