Antimicrobial Agents PDF

Antimicrobial Agents Terms Antimicrobial agent - kills or inhibits organisms natural: antibiotic semisynthetic: chemically modified antibiotics synthetic: man-made Antibiotics substances produced by bacteria and fungi that inhibit the growth of other organisms Terms An...

Antimicrobial Agents Terms Antimicrobial agent - kills or inhibits organisms natural: antibiotic semisynthetic: chemically modified antibiotics synthetic: man-made Antibiotics substances produced by bacteria and fungi that inhibit the growth of other organisms Terms Antibacterial agent Anitmicrobial agents that affect bacteria Chemotherapeutic agent Substance used to treat disease Includes antimicrobial and anticancer drugs Bactericidal – “cide” means kill Agent kills bacteria Bacteriostatic – “static” means no change Agent inhibits bacteria Terms Spectrum of activity Narrow spectrum – only certain groups covered Broad spectrum – gram pos and gram neg coverage Mechanism of action way agent harms organism Drug Combinations Additive effect is sum of activity of individual antimicrobial agents: 1+2=3 Synergism effect is amplified: 1+2=4 Antagonism one agent interferes with activity of other: 1+2=1 Mechanisms of Action Inhibition of bacterial cell wall synthesis Interference of plasma membrane Inhibition of folate synthesis Interference of DNA replication Interference of DNA transcription Interference of mRNA translation 50s Inhibitors 30s Inhibitors Cell Wall Characteristics Not found in mammalian cells Cell wall characteristics affect antibiotics spectrum of activity Gram-positive thick peptidoglycan cell wall Gram-negative thin peptidoglycan surrounded by outer membrane Peptidoglycan is AKA murein layer Gram-Positive and Gram- Negative Cell Walls Gram-Positive and Gram- Negative Cell Walls Inner (cytoplasmic) membrane Osmotic barrier Outer membrane (gram-negatives only) Lipopolysaccharides Phospholipids Porins Peptidoglycan Biosynthesis Synthesis of precursors in cytoplasm Transport of precursors across cytoplasmic membrane Insertion of precursors into cell wall Transpeptidation and transglycolation http://www.microbelibrary.org/images/spencer/spencer_cellwall.html Cell Wall Inhibitors Beta-lactam antibiotics Penicillins Ampicillin, Oxacillin, Methicillin Cephalosporins (Cephems) 1st thru 4th generation Cephalexin, cefotaxime, ceftriaxone Cell Wall Inhibitors Beta-lactam antibiotics Monobactams Aztreonam Cabapenems Imipenem, doripenem, ertapenem Broad and narrow spectrum Beta-Lactams Cell Wall Inhibitors Bind to penicillin-binding-proteins (PBPs) PBPs are transpeptidase/transglycosylase enzymes Beta-Lactams β -Lactam and β -Lactamase inhibitor combinations Amoxicillin and Clavulanic Acid (Augmentin) Clavulanic Acid or Clavulanate (β -Lactamase inhibitor) binds to β -Lactamases This binding prevents inactivation of β -Lactam drug (amoxicillin) Cell Wall Inhibitors Glycopeptides Vancomycin Dalbavancin Inhibit peptidoglycan synthesis Bind to terminal D-ala-D-ala Active vs. GP only Active vs. MRSA, C. difficile Large molecules can’t enter GN Other Polymyxins Plasma membrane disruption Active against gram-negative bacteria Nitrofurantoin Treat UTIs Folate Synthesis Folic acid pathway provides precursors for DNA synthesis Two key enzymes in pathway Dihydropteroate synthase Dihydrofolate reductase Folate Synthesis Inhibition Sulfamethoxazole and trimethoprim Broad spectrum Sulfonamides Sulfamethoxazole Mechanism of action: inhibits folic acid synthesis (DNA synthesis) Humans do not synthesize folic acid! Competitively binds PABA an essential component of metabolism For UTIs, enteric infections DNA and RNA DNA Replication Inhibition Enzymes required for DNA replication Topoisomerases DNA gyrases Quinolones/Fluoroquinolones Ciprofloxacin, levofloxacin, gemifloxacin Inhibit topoisomerase IV and/or DNA gyrase Broad spectrum, including P. aeruginosa DNA Transcription Inhibition Transcription mediated by RNA polymerase Rifampin binds to RNA polymerase Blocks RNA chain elongation Used to treat mycobacteria, staph, strep, enterococci mRNA Translation 30S and 50S ribosomal subunits bind to mRNA Protein Synthesis Inhibition Aminoglycosides Gentamicin, tobramycin, amikacin Inhibit 30S ribosomal subunit Active against GP and GN but not anaerobes Ototoxicity and nephrotoxicity Protein Synthesis Inhibition Tetracyclines Doxycycline, minocycline, tetracycline Inhibit 30S ribosomal subunit Broad spectrum, including mycoplasma and chlamydia Protein Synthesis Inhibition Macrolides Erythromycin, clarithromycin, azithromycin Inhibit 50S ribosomal subunit Broad spectrum Lincosamides (clindamycin) and Streptogramins (dalfopristin/quinupristin) Inhibit 50S ribosomal subunit Broad spectrum MLS group Protein Synthesis Inhibition Oxazolidinone (linezolid) Glycylcycline (tigecycline) Chloramphenicol Toxicity – aplastic anemia Mycobacterial Chemotherapy Isoniazid (INH) inhibits synthesis of mycolic acid (cell wall) Rifampin blocks RNA polymerase Ethambutol ? inhibits cell wall synthesis Streptomycin inhibits protein synthesis Mycobacterial Chemotherapy Therapeutic Considerations mutations are common (drug resistance is high) multiple drugs used to kill and prevent MDR- TB Mechanisms of Resistance Intrinsic Resistance resulting from normal genetic, structural, or physiologic state of the microorganism Naturally occurring Acquired Resistance resulting from altered cellular physiology and structure due to genetic changes Transfer or mutation to genetic code Mechanisms of Resistance Decreased uptake or accumulation Impermeability Efflux Biofilms Enzymatic inactivation or modification Target site modification Acquisition of new target Pathway bypass Mechanisms of Resistance Mechanisms of Resistance Impermeability of cell wall LPS and porins restrict entry of antimicrobials All GN resistant to vancomycin Can not cross outer membrane Efflux Transporters that remove toxic substances from bacterial cell Broad-spectrum resistance Mechanisms of Resistance Biofilms Bacteria in polysaccharide matrix Highly drug resistant (persister cells) Enzymatic inactivation or degradation Modification of aminoglycosides Inactivation by beta-lactamases Beta-Lactamases Hydrolyze the beta-lactam ring Produced by both GP and GN organisms β -lactamases vary in spectrum Most common β -lactamase enzyme is penicillinase Virtually all GN bacteria are intrinsically resistant via β -lactamase production Staph are most common GP producers Beta-Lactamases Cephalosporins were created to be forever resistant to all β -lactamase enzymes thus extending their spectrum of use Bacteria fought back and created extended spectrum beta-lactamases ESBLs β-lactamase enzymes that can inactivate all penicillins and cephalosporins Mechanisms of Resistance Target site modification: drug binds poorly (or not at all) to target site Altering of PBP decreases affinity for beta- lactam drugs Mutations to topoisomerase IV and DNA gyrase increases resistance to quinolones Addition of methylase to ribosome decreases binding of MLS antimicrobials Vancomycin-resistant enterococci altered cell wall Mechanisms of Resistance Vancomycin resistance vanA, vanB, vanC genes vanA on plasmid = transmissible Van-R enterococci transfer resistance to staph vancomycin intermediate S. aureus (VISA) and vancomycin resistant S. aureus (VRSA) have been isolated Mechanisms of Resistance Acquisition of new target site mecA gene Found in MRSA Encodes for a low affinity PBP Resistant to all penicillins Found in mobile SCCmec cassette Mechanisms of Resistance Bypass mechanisms Organism circumvents consequences of antimicrobial action Anaerobes are intrinsically resistant to aminoglycosides Lack of oxidative electron transport system Required for uptake of aminoglycosides Dissemination of Antimicrobial Resistance Transformation, transduction, conjugation Plasmids Extrachromosomal, circular, replicating DNA Transposons and Integrons Mobile DNA elements Resistance Expression Constitutive organism constantly expressing resistance mechanism Inducible resistance only when exposed to agent Constitutive-inducible constant expression at low levels Resistance Expression Homogenous entire bacterial population expressing resistance Heterogeneous some bacteria in population express resistance MRSA Chlamydia Introduction Three species cause human diseases Chlamydia trachomatis Chlamydia pneumoniae Chlamydia psittaci Differ from other bacteria Do not grow on nonliving media Sensitive to interferon Obligate intracellular parasites Characteristics Obligate intracellular parasite dependent on host for adenosine 5’- triphosphate (ATP) lack energy metabolism grow & multiply only inside animal or host epithelial cells Replication Cycle Elementary body Infectious Reticulate body non-infectious Replication Cycle Elementary body infects host cell EB organize into reticulate bodies and multiple After ~ 24 hrs, RB → EB Host cell ruptures and releases EB Replication Cycle Antigens Outer membrane similar to GN, but no peptidoglycan Components Lipopolysaccharide (LPS) Present in all Chlamydia species Major outer membrane protein (MOMP) Specific for each species and subspecies Species subdivided into serotypes (serovars) based on MOMP Chlamydia trachomatis Three biovars Trachoma Lymphogranuloma venereum (LGV) Mouse pneumonitis Each biovar contains different serovars C. trachomatis trachoma biovar Trachoma biovar contains serovars A - K Serovars A, B, Ba, C Cause trachoma eye infection Chronic eye infection Leading cause of blindness in the world Found near equator Causes scarring and abrasion of cornea C. trachomatis trachoma biovar Serovars D – K Inclusion conjuntivitis eye infection adults and newborns can colonize nasopharynx (lead to pneumonia) and genital tract Milder eye infections C. trachomatis trachoma biovar Serovars D – K Urogenital infections nongonococcal urethritis, epididymitis prostatitis in men pelvic inflammatory disease in females Most common bacterial STD Over 1M cases in 2007 C. trachomatis LGV biovar Serovars L1, L2, L2a, L2b, L3 TD Invasive urogenital disease Inguinal and anorectoal symptoms Enter lymph nodes near genital tract Inflammation bubos Seen in tropics, subtropics Newborn Chlamydia trachomatis Passed to newborn via birth canal Conjunctivitis, nasopharygeal infection, or pneumonia Infants given prophylatic eye drops for GC/Chlamydia Assumed to be the cause of all pneumonia in infants < 6 months, unless proven otherwise Specimens MUST contain host epithelial cells intracellular parasite Swab specimen avoid wooden sticks (toxic) endocervical, urethral, conjunctival Aspirates of lymph nodes or bobos (LGV) Lower RT secretions (C. pneumoniae or C. psittaci) Direct Microscopic Exam Direct specimen smears QC for presence of epithelial cells Rapid diagnosis of neonatal inclusion conjunctivitis and trachoma Sensitivity 95% Not sensitive for urogenital specimens Direct Microscopic Exam Stain cells/specimen with Giemsa stain purple inclusion bodies Iodine stains glycogen around EB detects only C. trachomatis  Direct Microscopic Exam Chlamydia inclusion bodies Chlamydia glycogen stained on Giemsa stain brown on iodine stain Direct Microscopic Exam Direct fluorescent antibody (DFA) fluorescein-labeled anti-Chlamydia antibodies genus specific LPS and species specific MOMP most sensitive method Cell Cultures Approximately 80% sensitive Viral culture techniques required living cells/tissue cultures Avoid viral transport media contain antimicrobial agents Technically demanding Cell Cultures Growth in cell lines Cell culture Shell vial system Demonstrate inclusions in culture cells with giemsa, iodine, or fluorescent stain Non-Culture Detection Methods Enzyme immunoassay (EIA) - detects LPS or MOMP antigen false positive can occur not as sensitive as culture Serology – limited and problematic infections localized antibodies can be from previous infections Non-Culture Detection Methods Nucleic acid assays - detects both Chlamydia and GC DNA probe amplification methods (PCR) very sensitive and specific – new gold standard, problems with culture Use of Culture and Non- Culture Methods Resources Patient population Cultures: ~100% specific, 80% sensitive time consuming, experience Non-culture: presumptive if positive confirmed by culture or another non-culture method nucleic probes: new gold standard ? Chlamydia pneumoniae Mild or asymptomatic Affects young adults Prolonged pharyngitis followed by bronchitis or pneumonia Potential risk factor for Guillain-Barré syndrome Asthma Cardiovascular disease C. pneumoniae Common infection in US Detected using serologic methods Same culture techniques  Chlamydia psittaci Cause ornithosis psittacosis or parrot fever zoonotic infection Occupational hazard for pet bird handlers and poultry workers Rare in US C. psittaci Acute lower respiratory infection Cultures not sensitive BSL3 Diagnosed serologically Antimicrobial Susceptibility Testing and Therapy Chapter 13 Ideal Antibiotic Selective toxicity – exerts effects on the agent of disease w/o harming patient Treats diseases caused by GP and GN (broad spectrum) Host does not develop hypersensitivity or allergic reaction The agent penetrates into tissues and crosses the blood-brain barrier (CNS infections) The cost is minimal Susceptibility Terms Minimum inhibitory concentration (MIC) lowest concentration of antibiotic that visibly inhibits growth of an organism Minimum bactericidal concentration (MBC) lowest concentration results in the death or killing of >99.9% of an organism Susceptible or Sensitive – interpretive category that indicates an organism is inhibited based on amount of drug safely achieved in patient (µg/mL) Intermediate – interpretive category that indicates an organism may require a higher dose of antibiotic for a longer period of time to be inhibited Resistant – interpretive category that indicates an organism is not inhibited by the recommended dose or achievable level Susceptibility Standards Clinical and Laboratory Standards Institute (CLSI) formerly the National Committee for Clinical Laboratory Standards (NCCLS) develops interpretative standards for methods Testing categories susceptible (S) intermediate (I) resistant (R) Test Batteries Panel of 10 – 15 antimicrobial agents for routine testing against commonly isolated organisms Panels may be selected by GN, GP, urine Agents categorized by organism groups Group A = primary agents, reported first Group B = if group A agents ineffective or not able to administer Groups C = supplemental, report selectively Group U = urine only Preparation of Inoculum Requires pure culture, organisms inoculated into a broth medium Most important step of all susceptibility testing How to determine # of bacteria per mL? Compare to a standard of turbidity representing a known # of bacteria McFarland Standards 0.5, 1, 2, 3, … McFarland 0.5 = 1.5 x 108 CFU/mL Standardized Testing Methods Broth Dilution Method Antibiotic is suspended in a solution Agar Dilution Method Antibiotic is incorporated into the agar Diffusion Method Uses antibiotic impregnated disks or strips Special Methods Broth Dilution Tests Provides MIC Antibiotic powders Serial dilution in Mueller-Hinton broth Add 2 - 5% horse blood for strep Use Haemophilus Test Medium (HTM) for Haemophilus MH supplemented with hemin and NAD Final bacterial concentration = 5 x 105 CFU/mL Broth Dilution Tests Test methods Macrodilution tubes with 1 to 2 mL broth Microdilution microtiter trays (0.05 - 0.1 mL) Broth Dilution Tests Test includes internal controls growth control - positive growth (no antibiotics) sterility check - no inoculation - no growth Purity plate is an external control inoculum subcultured onto BAP agar to ensure it is a pure culture Purity plate, growth well, sterility well checked first Broth Dilution Macrodilution Broth Dilution Tests: MBC Performed in limited situations Aliquot removed from growth tube, inoculated onto BAP, incubated # organisms per mL calculated on each tube with no visible growth Colony count compared to inoculum colony count MBC = concentration killing 99.9% of organisms MBC = 1 log reduction in count Serum Bactericidal Test Schlichter test Tests patient serum (containing antibiotics) against infecting organism Incubate overnight and examine for growth Peak and trough specimens Trough: just before drug administered Peak: 30-90 minutes after dose Microdilution Similar to macrodilution Plastic microdilution panels inoculated to give MIC Each well holds a micro amount of antibiotic to be tested in varying concentrations Microdilution MIC for Cefazolin is 4 ug/ml MIC for Ampicillin is > 32ug/ml MIC for ciprofloxacin is ≤ 0.25 ug/ml Agar Dilution Tests Semi-quantitative Antimicrobial agents incorporated into agar medium Series of agar plates with different concentrations Standardized suspension inoculated Incubated and examined for growth Disk Diffusion Tests: Kirby Bauer Paper disks impregnated with antibiotic placed on inoculated plates Incubated at 35°C in ambient air and zone of inhibition measured Agent diffuses through agar in circle, inhibiting bacterial growth zone of inhibition Measure zone of inhibition Large zones of inhibition indicate more antimicrobial activity or greater diffusion No zone indicates complete resistance to the drug Kirby Bauer Disk Diffusion resistance inhibition Mueller-Hinton (MH) Agar Standard media in disk diffusion tests QC weekly with E. coli, S. aureus, P. aeruginosa Incubated for 18 hours at 35°C, ambient air Agar depth: 4 mm Agar depth too thin leads to increased zones and false susceptibility Agar depth too thick leads to decreased zones and false resistance Agar pH 7.2-7.4 Decreased pH can lead to decreased activity of aminoglycosides, erythromycin, and clindamycin and increased activity of tetracyclines Increased pH has the opposite effect Mueller-Hinton (MH) Agar Fixed cation concentration (Ca++ & Mg++) increased concentrations result in decreased activity of aminoglycosides against P. aeruginosa and decreased activity of tetracyclines decreased concentrations have the opposite effect Kirby Bauer Procedure Inoculation standardized suspension (0.5 McFarland) cotton swab streaked over plate in 3 directions Disk application within 15 minutes of inoculation Incubate 18 – 24 hours Measure zones against a dark background ruler or caliper Disk Diffusion Test Interpretation Size of zone dependent of disk concentration S, I, R based on size of zone Susceptible isolate: large zone and low MIC Resistant: small zone and high MIC Interpretations S, I, R of zone diameter are based on CLSI sensitivity tables for each antibiotic S, I, R determined by plotting zone diameters against MICs of a large # of isolates Correlation of Disk Diffusion and MIC Diffusion E-Test Agar plates inoculated in same manner as disk diffusion Plastic strip containing gradient of antimicrobial agent placed on surface and diffuses through agar Incubated and examined for elliptical zone Diffusion E-Test Test Performance Inoculum from overnight growth MHA plus sheep blood used for strep, Haemophilus test medium for Haemophilus spp. Incubated in 5-7% CO2 for 18-20 hours Chocolate MHA available Disks stored at proper temperature with desiccant Disks not expired Decreased potency Allow disks to warm to RT before use Reading and Interpretation Lawn of growth must be confluent Ignore tiny colonies at zone edge Ignore Proteus spp. Swarming Obvious colonies within clear zone should not be ignored Contamination Mixed culture Resistant subpopulation Special Tests β-lactamases, ESBLs, Carbapenemases Altered PBPs Inducible Macrolide Resistance Oxacillin Resistance Vancomycin Resistance High-Level Aminoglycoside Resistance Anaerobes β-Lactamase Tests β-Lactamase testing clinical applications detects beta-lactamase-mediated resistance of organisms to penicillinase-susceptible penicillins (penicillin, ampicillin) Rapid test does not require overnight incubation like MIC and disk diffussion β-lactamase testing needed for H. influenzae N. gonorrhoeae Morexella catarrhalis Staphylococcus spp. Bacteroides spp. β-Lactamase Test Methods Nitrocefin - chromogenic cephalosporin intact beta lactam ring → yellow color (negative) If a β -lactamase enzyme cleaves the beta Cefinase Disk lactam ring → red color (positive) most sensitive and common test Acidimetric phenol red pH indicator If β -lactamase splits penicillin → penicilloic acid (yellow=positive) Iodometric starch-iodine indicator β-Lactamase Haemophilus, Neisseria, Bacteroides spp., and Moraxella Constitutive expression Staphylococcus spp. Inducible expression May have to expose staph to inducing agent (β-lactam) to stimulate sufficient production for detection Enterobacteriaceae and Pseudomonas spp. produce many different types of β-lactamases Cannot predict resistance with nitrocefin ESBLs Typically seen in Klebsiella, Proteus, E. coli Test against cephalosporins and monobactam Cefpodoxime, Ceftazidime, Cefotaxime, Ceftriaxone Aztreonam Confirm by testing with β-lactamase inhibitor Clavulanic acid Antimicrobial activity restored Report ESBL-producers as resistant to all cephalosporins, penicillins, and monobactams Carbapenamases Initially identified in K. pneumoniae (KPC) Now in many Enterobacteriaceae Resistance to carbapenems Imipenem, Meropenem, Ertapenem Altered PBPs Detect penicillin resistance Use disk susceptibility test for pneumococci Oxacillin disk More accurate than penicillin disk Macrolide Resistance Isolate appears erythromycin resistant but clindamycin susceptible Inducible clindamycin resistance may be present Seen in staph and strep D test must be performed Place erythromycin and clindamycin disks on MH agar No inhibition zone around erythromycin Decreased clindamycin inhibition zone on side next to erythromycin Positive for inducible resistance Oxacillin-Resistant Staphylococci (MRSA) Oxacillin more reliable than methicillin Belong to same class of agents Penicillinase-resistant penicillins Isolate resistant to one = resistant to all Mec-A mediated resistance Staph are heteroresistant = difficult to detect Oxacillin-Resistant Staphylococci (MRSA) Modify testing conditions Supplement MH with 2% NaCl Incubate at < 35°C for 24 hours Any growth is considered significant Hazy zone – Clear zone – Heteroresistance Hyper β-lactamase producer Oxacillin-Resistant Staphylococci (MRSA) Oxacillin screen plate MH agar + 4% NaCL + 6 µg/mL oxacillin Prepare 0.5 MacFarland suspension Inoculate plate Incubate overnight Any growth is significant  Vancomycin Resistance MRSA isolates with resistance to vancomycin VISA and VRSA Recommended method of detection (staph or enterococci) Broth dilution Vancomycin agar screen (at 6 µg/mL) Heteroresistant vancomycin intermediate strains (hVISA) may be detected by macro E Test Uses a higher concentration of organisms in inoculum Vancomycin Resistance E. faecium more resistant than E. faecalis Intrinsically resistant E. gallinarium E. casseliflavus Intrinsic, high-level resistance Leuconostoc spp. Pediococcus spp. Lactobacillus spp. High-Level Aminoglycoside Resistance in Enterococci Enterococci may be treated with ampicillin or penicillin Bacteriostatic only Combined with aminoglycoside to achieve bactericidal effect Enterococci are intrinsically resistant to low levels of aminoglycosides Synergistic with cell wall active agent High-Level Aminoglycoside Resistance in Enterococci High-level aminoglycoside resistance due to enzymatic inactivation Detect using broth, agar, or disk Screening concentrations Broth = 500 to 1000 µg/mL Anaerobes Reference method is agar dilution Supplemented Brucella laked sheep blood agar Clinical lab may use broth microdilution Brucella broth with lysed horse blood Inoculum is higher, 1 x 106 CFU/mL Incubation longer, 48 hours Automated Systems Optical methods to detect susceptibility endpoints Turbidometric Hydrolysis of fluorometric growth substrate Analyses in shorter period ( 5 to 15 hours) Use microprocessors Also used to identify GN and GP organisms Automated Systems BD Phoenix Microscan Walkaway Automated Systems Trek Sensititre Vitek Vitek QC Interpretations determined by CLSI Reference strains American Type Culture Collection (ATCC) Defined susceptibility/resistance patterns Range of endpoints (S, I, R) Endpoint is on-scale Acceptable results defined S. aureus ATCC 29213 amikacin MIC range 1-4 µg/mL QC Testing each day patient testing performed Reduced to weekly after acceptable performance with QC strains Acceptable results for each drug/bug combo for 20 or 30 consecutive test days Always performed with new lot numbers Supplemental QC Periodic testing for specific combos MRSA with heteroresistance Ampicillin-resistant E. cloacae Antibiograms Verify patient results Overall antimicrobial susceptibility profile of an organism E. coli susceptible to most drugs S. maltophilia resistant to most drugs Predictor Drugs Staphylococci resistant to oxacillin Report R to all beta-lactam drugs Enterococci with high-level aminoglycoside resistance Report R to all aminoglycosides Enterococci resistant to ampicillin Report R to all penicillin derivatives Susceptibility Testing Full Susceptibility Testing Required: Staphylococci S. pneumoniae Viridans strep (if from a normally sterile site) Enterococci Enterobacteriaceae Pseudomonas aeruginosa Acinetobacter spp.  Susceptibility Testing Testing occasionally required (β -lactamase testing) H. influenzae N. gonorrhoeae Morexella catarrhalis Anaerobes Rarely required: organisms are susceptible to beta- lactam drugs Beta hemolytic streptococci Neisseria meningitidis Listeria monocytogenes Miscellaneous Bacteria 1  Miscellaneous Bacteria Mycoplasma Ureaplasma Rickettsia Ehrlichia Anaplasma Coxiella burnetii Miscellaneous GNR 2 Mycoplasma & Ureaplasma Belong to class Mollicutes Size of a large virus Smallest self-replicating microorganisms Colonize mucous membrane of RT and UGT Humans, animals Transmission Sexual contact Mother to child Respiratory secretions 3 Mycoplasma & Ureaplasma No cell wall Do not gram stain Pleomorphic Utilize sterols to create membrane support Resistant to cell wall antibiotics Penicillin and cephalosporins Slow growing Fastidious Cell culture 4 Mycoplasma pneumoniae Primary atypical pneumonia walking pneumonia teenagers and young adults Bronchitis, pharyngitis 5 Mycoplasma hominis Colonizes urogenital tract Opportunistic pathogen Upper urogenital tract infections PID, salpingitis, pyelonephritis 6 Other Mycoplasma Species M. genitalium NGU, PID, cervicitis, endometriosis,sterility M. fermentans Opportunistic respiratroy pathogen M. penetrans Urine of men with HIV M. salivarium Synovial fluid of RA patients 7 Ureaplasma urealyticum Colonize urogenital tract Non-chlamydial, non-gonococcal urethritis in men Associated with upper genital tract infections in women Hydrolyzes urea 8 Mycoplasma & Ureaplasma Can be transmitted to newborns at delivery Set up culture for these organisms if meningitis suspected, but Negative gram stain and culture 9 Specimen Collection and Transport Body fluids, wound aspirates, tissue samples NP, cervical, vaginal swabs Inoculate at bedside Extremely sensitive to drying and heat Transport media Tryptic soy agar with penicillin SP4 (sucrose phosphate buffer, serum) Plate immediately or store at -70°C 10 Cultures Special media: A7, E agar, U broth Enriched with sterols and penicillin 1 week for most 3-4 weeks for M. pneumoniae Mycoplasma: fried-egg colony morphology Ureaplasma: small colonies 11 Mycoplasma on Enriched Media 12 Mycoplasma and Ureaplasma Microscopic image of Mycoplasma hominis (ATCC® 23114) and Ureaplasma urealyticum (ATCC® 27618) colonies growing on SP4 Agar with Glucose (Cat. no. G21). Incubated aerobically for 72 hours at 35 deg. C. All colonies are mycoplasma except the dark one, which is ureaplasma. 13 Mycoplasma ID Recovery from culture difficult ~ 40% sensitivity Detect serum IgG and IgM antibodies Demonstrate a fourfold rise in cold agglutinin titer Complement fixation (more specific) titers Enzyme immunoassay Microimmunofluorescence Radiography for Dx of M. pneumoniae 14 Rickettiaceae and Similar Organisms Arthropod-borne Obligate intracellular parasites GN coccobacilli Rickettsia spp. Ehrlichia spp. Coxiella burnetii 15 Rickettsia Predilection for endothelium of blood vessels Never grown in cell-free media Pleomorphic GN coccobacilli Nonmotile Two groups Spotted fever group Typhus group 16 Rickettsial Spotted-Fever Group R. rickettsiae Rocky Mountain Spotted Fever Vector is ticks Flulike symptoms Characteristic rash on palms of hands and soles of feet May disseminate to blood vessels of lungs, brain, heart Untreated mortality rate ~20% 17 Rocky Mountain Spotted Fever 18 Rocky Mountain Spotted Fever 19 Rickettsial Spotted-Fever Group R. akari Rickettsialpox Vector is mouse mites Seen in Eastern US Similar to RMSF but milder except Rash of face, body, extremities Not on palm and soles 20 Rickettsialpox 21 Rickettsial Typhus Group R. typhi Endemic typhus or murine typhus Vector is rat fleas Flulike symptoms and +/- rash 22 Rickettsial Typhus Group R. prowazekii Epidemic louse-borne typhus War and natural disasters Vector is human body and squirrel louse Flulike symptoms Rash on palms, soles, face Untreated mortality rate ~ 40% Brill-Zinsser disease Reactivation of infection 23 Orientia tsutsugamushi Formerly Rickettsia Causes scrub typhus Vector is rat chigger Flulike symptoms and rash 24 Safety Highly infectious Handle rickettsia in BSL3 lab BSL2 OK for separating serum from clotted blood 25 Rickettsial Diagnosis Immunohistochemical staining for Dx Serology confirms Dx IFA – immunofluorescence assay Weil-Felix agglutination reaction (nonspecific) serum of patients reacted with strains of Proteus (OX- 19, OX-2, OX-K) R. rickettsiae positive for OX-19 and OX-2, negative for OX-K Commercially available agglutination kit 26  Immunohistochemical Staining Bacteria in the endothelial and inflammatory cells of a blood vessel in the dermis (arrow). 27 Ehrlichia and Anaplasma Pleomorphic GN coccobacilli Obligate intracellular parasite Developmental cycle similar to Chlamydia EB infectious form Replicates in phagocytes Form inclusions called morulae 28 Ehrlichia and Anaplasma Morulae 29 Ehrlichia chaffeensis Causes human monocytic ehrlichiosis (HME) Associated with tick bites (Southern US) Fever, headache, muscle pain, malaise May have leukopenia, neutropenia, thrombocytopenia and elevated liver enzymes Can be severe 30 Anaplasma phagocytophilum Formerly Ehrlichia Human granulocytic ehrlichiosis (HGE) Infects neutrophils Tick bites (Northern US) Similar symptoms to Ehrlichia 31 Ehrlichia and Anaplasma ID Peripheral blood smear Wright, Giemsa stain or histologic stains Nucleic acid amplification testing PCR: 90% sensitive, 100% specific Serology (retrospective) 32 Coxiella burnetii Obligate intracellular GN coccobacilli Causes Q fever Zoonosis – cattle, sheep, goats Found in urine, feces, milk, birth products Remain in soil for years (spore-like lifecycle) 33 Coxiella burnetii Transmitted by inhaling infectious aerosols Infects lungs → systemic infection Flulike symptoms, liver enzymes, thrombocytopenia Highly contagious → BSL 3 Complication: endocarditis, death (fatal disease) Dx by direct immunofluorescence, serology, PCR 34 Miscellaneous GNR Bartonella spp. Spirillum minus Klebsiella granulomatis Chryseobacterium Chromobacterium violaceum 35 Barttonella quintana Trench fever (5-day fever) - homeless Bartonella spp. multiply in RBC and endothelial cells Bacillary angiomatosis (BA) proliferation of blood vessels forming a mass immunocompromised individuals (HIV) Bacteremia, endocarditis Transmitted by body lice 36 Barttonella henselae Cat-scratch disease lesion and fever, recovery in 2 - 4 months Transmitted by cat fleas Bacillary angiomatosis, bacteremia, endocarditis Detected by silver stain 37 Presumptive Identification Slow growth on CHOC and BAP in CO2 Small GNR Oxidase and catalase negative Histopathology Organisms in tissue Warthin-Starry stain 38 Spirillum minus Helically coiled GNR Causes rat-bite fever (sodoku) Transmitted by rat bites and scratches Similar to Streptobacillus moniliformis Doesn’t grow on artificial media Dark field or stain with Giemsa 2 - 3 spirals and bipolar polytrichous tufts of flagella Cultured by injecting specimen in rats and recovering 39 Spirillum minus 40 Klebsiella granulomatis Formerly Calymmatobacterium granulomatis GNR Causes granuloma inguinale or donovanosis Ulcerative STD Diagnosis Detect “Donovan bodies” in Giemsa-stained tissue smears Encapsulated GNR in mononuclear cells Safety pin appearance Cultivation is difficult 41 Chryseobacterium Formerly Flavobacterium C. indologenes, C. meningosepticum Ubiquitous in soil and water, not normal flora Nosocomial infections GNR that is a weak to nonfermenter Nonmotile, pigmented, fruity odor DNase, oxidase, indole positive 43 Chromobacterium violaceum Found in soil & water Produce violacein (purple or violet pigments) when exposed to O2 Can cause wound infections and bacteremia Facultative anaerobe Grow on BAP, CHOC, MAC Grow at 35°C or 42°C, prefers 25°C enhance pigment production 45 Chromobacterium violaceum Cyanide odor colonies produces hydrogen cyanide Ferment glucose Motile Catalase positive Variable oxidase - harder to read due to color of pigments 47