Medical Microbiology Quiz

Antimicrobial Agents

• 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
• Antibacterial agent - Antimicrobial agents that affect bacteria
• Chemotherapeutic agent - substance used to treat disease
• Bactericidal - "cide" means kill; agent kills bacteria
• Bacteriostatic - "static" means no change; agent inhibits bacteria

Spectrum of Activity

• Narrow spectrum - only certain groups covered
• Broad spectrum - gram positive and gram negative coverage

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

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)

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

• Diagrams showing the structural differences, highlighting the cell wall layers, and cytoplasmic membranes of gram-positive and gram-negative bacteria.

Peptidoglycan Biosynthesis

• Synthesis of precursors in cytoplasm
• Transport of precursors across cytoplasmic membrane
• Insertion of precursors into cell wall
• Transpeptidation and transglycolation

Cell Wall Inhibitors

• Beta-lactam antibiotics
  • Penicillins (Ampicillin, Oxacillin, Methicillin)
  • Cephalosporins (Cephalexin, cefotaxime, ceftriaxone)
  • Monobactams (Aztreonam)
  • Carbapenems (Imipenem, doripenem, ertapenem)
• Glycopeptides
  • Vancomycin
  • Dalbavancin

Other

• Polymyxins - plasma membrane disruption
• Active against gram-negative bacteria
• Nitrofurantoin - treats UTIs

Folate Synthesis

• Folic acid pathway provides precursors for DNA synthesis
• Dihydropteroate synthase
• Dihydrofolate reductase
• 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

• Transcription mediated by RNA polymerase
• RNA polymerase is targeted by Rifampin
• blocks RNA chain elongation treating mycobacteria, staph, strep, enterococci

DNA Replication Inhibition

• Enzymes required for DNA replication: Topoisomerases, DNA gyrases
• Quinolones/Fluoroquinolones (Ciprofloxacin, levofloxacin, gemifloxacin) – inhibits topoisomerase IV and/or DNA gyrase, broad spectrum, including P. aeruginosa

mRNA Translation

• 30S and 50S ribosomal subunits bind to mRNA.

Protein Synthesis Inhibition

• Aminoglycosides (Gentamicin, tobramycin, amikacin) – inhibits 30S ribosomal subunit, active against GP and GN but not anaerobes, ototoxicity and nephrotoxicity
• Tetracyclines (Doxycycline, minocycline, tetracycline) – inhibits 30S ribosomal subunit, broad spectrum including mycoplasma and chlamydia
• Macrolides (Erythromycin, clarithromycin, azithromycin) – inhibits 50S ribosomal subunit, broad spectrum.
• Lincosamides (clindamycin) and Streptogramins (dalfopristin/quinupristin) – inhibits 50S ribosomal subunit, broad spectrum
• 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 are used to kill and prevent MDR-TB (Multi-drug resistant Tuberculosis)

Mechanisms of Resistance

• Intrinsic - normal genetic, structural, or physiologic state
• Acquired - altered cellular physiology, genetic changes, or mutations in genetic code

Mechanisms of Resistance (continued)

• Decreased uptake or accumulation (impermeability, efflux, biofilms)
• Enzymatic inactivation or modification (target site modification)
  • Acquisition of new target
  • Pathway bypass
• Impermeability of cell wall
• Efflux
• Biofilms
• Modification of aminoglycosides
• Inactivation by beta-lactamases

Beta-Lactamases

• Hydrolyze the beta-lactam ring.
• Produced by both gram-positive and gram-negative organisms
• Most common beta-lactamase enzyme is penicillinase.
• Virtually all gram-negative bacteria are intrinsically resistant via beta-lactamase production
• Staph are most common gram-positive producers

Beta-Lactamases (continued)

• Cephalosporins were created to be resistant to all beta-lactamases
• Beta-lactamases vary in spectrum of use
• Extended spectrum beta-lactamases (ESBLs) - beta-lactamase enzymes that inactivate all penicillins and cephalosporins

Mechanisms of Resistance (continued)

• 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 increase resistance to quinolones.
• Addition of methylase to ribosome decreases binding of MLS antimicrobials
• Vancomycin-resistant enterococci altered cell wall

Mechanisms of Resistance (continued)

• Vancomycin resistance (vanA, vanB, vanC genes)
• vanA on plasmid is transmissible
• Van-resistant enterococci transfer resistance to staph - vancomycin intermediate S. aureus (VISA) and vancomycin resistant S. aureus (VRSA)
• Acquisition of new target site (mecA gene; found in MRSA; encodes for low affinity PBP; resistant to all penicillins).
• Bypass mechanisms - organism circumvents consequences of antimicrobial action.
• Anaerobes are intrinsically resistant to aminoglycosides because they lack the oxidative electron transport system required for uptake.

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
• Homogenous - entire bacterial population expressing resistance.
• Heterogeneous - some bacteria in population express resistance

Chlamydia

• Three species cause human diseases: Chlamydia trachomatis, Chlamydia pneumoniae, and Chlamydia psittaci
• Differ from other bacteria by not growing on non-living media, sensitivity to interferon, and being obligate intracellular parasites.
• Characteristics: obligate intracellular parasite, dependent on host for adenosine-5'-triphosphate (ATP), lack energy metabolism, grow and multiply only inside animal or host epithelial cells
• Replication cycle: elementary body (infectious), Reticulate body (non-infectious)
• Antigens: outer membrane similar to Gram-negative bacteria, but no peptidoglycan. Components: lipopolysaccharide (LPS), major outer membrane protein (MOMP), specific for each species and subspecies, species subdivided into serotypes (serovars) based on MOMP
• Chlamydia trachomatis: 3 biovars (Trachoma, Lymphogranuloma venereum (LGV), Mouse pneumonitis), each biovar contains different serovars
• Serovars D-K responsible for: inclusion conjunctivitis, eye infections in adults and newborns, colonization in the nasopharynx (leading to pneumonia) and in the genital tract. Urogenital infections, non-gonococcal urethritis, epididymitis, prostatitis in men, pelvic inflammatory disease (PID) in females
• LGV biovar - serovars L1, L2, L2a, L2b, L3. Invasive urogenital disease, inguinal and anorectal symptoms, enter lymph nodes near genital tract.
• Newborn Chlamydia trachomatis: transmitted via birth canal, can cause conjunctivitis, nasopharyngeal infection, or pneumonia.
• Specimens: MUST contain host epithelial cells, avoid wooden sticks (toxic), and may include endocervical, urethral, conjunctival samples
  • lower respiratory tract (RT) secretions for Ch. pneumoniae and Ch. psittaci

Chlamydia trachomatis - Trachoma biovar

• Serovars A-K - cause trachoma eye infection. Chronic eye infection. Leading cause of blindness in world. Found near equator. Causes scarring and abrasion of cornea.

Chlamydia trachomatis - Trachoma biovar

• Serovars D-K - inclusion conjunctivitis in adults and newborns. Can colonize nasopharynx (lead to pneumonia) and genital tract. Milder eye infections

Chlamydia trachomatis - LGV biovar - Serovars L1, L2, L2a, L2b, L3

• Invasive urogenital disease, Inguinal and anorectal symptoms, Enter lymph nodes near genital tract, Inflammation. Seen in tropics and subtropics

Chlamydia pneumoniae

• Common infection in US, detected using serological methods. Same culture techniques
• Mild or asymptomatic
• Affects young adults
• Prolonged pharyngitis followed by bronchitis or pneumonia
• Potential risk factor for Guillain-Barré syndrome, asthma, and cardiovascular disease

Chlamydia psittaci

• Causes ornithosis (psittacosis or parrot fever), zoonotic infection
• Occupational hazard for pet bird handlers and poultry workers, rare in US
• Acute lower respiratory infection
• Cultures not sensitive (BSL3)
• Diagnosed serologically

Antimicrobial Susceptibility Testing and Therapy

• Ideal antibiotic: selective toxicity to the pathogenic agent w/o harming the patient, treats diseases caused by GP and GN, host does not develop hypersensitivity/allergic reaction, penetrates tissues/blood-brain barrier, and cost is minimal
• Susceptibility Terms: MIC - lowest concentration visibly inhibiting organism growth, MBC - lowest concentration killing >99.9% of organism, Susceptible/Sensitive, Intermediate, Resistant
• Susceptibility Standards: Clinical and Laboratory Standards Institute (CLSI) or National Committee for Clinical Laboratory Standards (NCCLS) develops standards for testing methods (S, I, R)
• Test Batteries: Panel of antimicrobial agents for routine testing against commonly isolated organisms (selected by organism group e.g., GN, GP, or urine)
• Group A- primary agents, reported first
• Group B- if group A agents are ineffective or cannot be administered
• Group C- supplemental
• Group U- urine only
• Preparation of Inoculum: requires a pure culture, most important step of susceptibility testing. Determine # bacteria per mL, compare to standard of turbidity (e.g., McFarland standards for known # bacteria)

Standardized Testing Methods

• Broth Dilution Method - antibiotic suspended in a solution
• Agar Dilution Method - antibiotic incorporated into the agar
• Diffusion Method - antibiotic impregnated disks or strips
• Special Methods

Broth Dilution Tests

• Provides MIC, Serial dilution in Mueller-Hinton broth, add 2-5% horse blood (for strep) or Haemophilus Test Medium (HTM), MH supplemented with hemin and NAD (for Haemophilus spp), Final bacterial concentration is 5 x 10$^5$ CFU/mL.
• Test methods: Macrodilution (tubes with 1 to 2 mL broth), Microdilution (microtiter trays 0.05-0.1 mL)
• Internal controls: Growth control (positive growth no antibiotics), sterility check (no inoculation, no growth)
• Purity plate: inoculated onto BAP agar, Growth well, sterility well checked first

Broth Dilution Tests: MBC

• Performed in limited situations, Aliquot removed from growth tube, inoculated onto BAP, incubated, Organisms per mL calculated for each tube with no visible growth
• Colony count compared to inoculum colony count
• MBC = concentration killing 99.9% of organisms - 1 log reduction in count

Serum Bactericidal Test

• Schlichter test, Tests patient serum with antibiotics against infecting organism, Incubate overnight, 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 for MIC, Each well holds a micro amount of antibiotic in varying concentrations

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, agent diffuses through agar, Measure zone of inhibition.
• Large zones = more antimicrobial activity
• No zone = complete resistance

Mueller-Hinton (MH) Agar

• Standard media for 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; Thin depth = increased zones, false susceptibility; thick depth = decreased zones, false resistance
• pH 7.2-7.4; decreased pH = decreased activity of aminoglycosides, erythromycin, and clindamycin, Increased activity of tetracyclines
• 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
• Disk Diffusion Test Interpretation: Size of Zone dependent of disk concentration, Susceptible isolate: large zone and low MIC; Resistant: small zone and high MIC

Diffusion E-test

• Agar plates inoculated; Plastic strip with antimicrobial gradient placed on agar, Diffuses through agar
• Incubated and examined for elliptical zone

Test Performance

• Inoculum from overnight growth - MHA with sheep blood for strep, Haemophilus
• Incubated in 5-7% CO2, 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 colony within clear zone = ignored
• Can be contamination or mixed culture, or resistant subpopulation

Special Tests

• Beta-lactamases, ESBLs, Carbapenemases
• Altered PBPs
• Inducible Macrolide Resistance
• Oxacillin Resistance
• Vancomycin Resistance
• High-Level Aminoglycoside Resistance
• Anaerobes

Beta-Lactamase Tests

• Detects beta-lactamase-mediated resistance to organisms - rapidly tests.
• Needed for H. influenzae, N. gonorrhoeae, Moraxella catarrhalis, Staphylococcus spp, Bacteroides spp.

Beta-Lactamase Test Methods

• Nitrocefin - chromogenic cephalosporin (intact beta-lactam = yellow; broken beta-lactam = red).
• Acidimetric - phenol red pH indicator (penicilloic acid = yellow = positive)

Beta-Lactamase

• Haemophilus, Neisseria, Bacteroides spp, Moraxella
• Constitutive expression
• Staphylococcus spp
• Inducible expression
• May need to expose staph to inducing agent (beta-lactam) to stimulate production
• Enterobacteriaceae & Pseudomonas spp. many different types of beta-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 via beta-lactamase inhibitor (Clavulanic acid),Restored Antimicrobial activity
• 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

• Detects penicillin resistance
• Use disk susceptibility and oxacillin disk (more accurate than penicillin disk)

Macrolide Resistance

• Isolate appears erythromycin-resistant but clindamycin-resistant may be present
• Seen in staph and strep
• Performed in MH agar
• Decreased clindamycin inhibition zone, 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 =heteroresistant = difficult to detect
• Modify testing conditions: Supplement MH with 2% NaCl, Incubate at < 35°C for 24 hours
• Any growth is significant
• Clear zone (hyper beta-lactamase producer), hazy zone (heteroresistance)

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), Broth dilution, Vancomycin agar screen (at 6 µg/mL), Detected by macro E Test (uses a higher concentration of organisms in inoculum)
• E. faecium more resistant than E. faecalis, Intrinsically resistant E.gallinarium, E. casseliflavus
• Intrinsic, high-level resistance in Leuconostoc spp., Pediococcus spp.,and 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, Intrinsically resistant to low levels of aminoglycosides, Synergistic with cell wall active agent
• High-level 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 10$^6$ 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, Used to identify GN and GP organisms. Machines: BD Phoenix, Microscan Walkaway, Trek Sensititre, and 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 (e.g., S. aureus ATCC 29213 amikacin MIC range 1-4 µg/mL)
• Testing each day, reduced to weekly; 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 (e.g., MRSA with heteroresistance, Ampicillin-resistant E. cloacae), Verify patient results, Overall antimicrobial susceptibility profile of an organism (e.g., E. coli; S. maltophilia)

Predictor Drugs

• Staphylococci resistant to oxacillin (report resistance to all beta-lactam drugs)
• Enterococci with high-level aminoglycoside resistance (report as resistant to all aminoglycosides)
• Enterococci resistant to ampicillin (report resistance 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, and Acinetobacter spp.
• Testing occasionally required (beta-lactamase testing): H. influenzae, N. gonorrhoeae, Morexella catarrhalis, Anaerobes, Beta-hemolytic streptococci, Neisseria meningitidis, and Listeria monocytogenes

Miscellaneous Bacteria

• Mycoplasma, Ureaplasma, Rickettsia, Ehrlichia, Coxiella burnetii
  • Mycoplasma (Mollicutes) characteristics, no cell wall, do not gram stain, pleomorphic, resistant to penicillin and cephalosporins, slow growing, fastidious, cell culture required, atypical pneumoniae & other infections.
• Ureaplasma (Mollicutes) characteristics, colonized urogenital tract, non-chlamydial, non-gonococcal urethritis in men, upper genital tract infections in women, hydrolyzes urea
• Rickettsia (arthropod-borne obligate intracellular parasites), GN coccobacilli, predilection for endothelium of blood vessels; never grown in cell-free media. Types: R. rickettsiae, R. akari, R. typhi, R. prowazekii, and Orientia tsutsugamushi - each causes different diseases.
• Ehrlichia and Anaplasma (pleomorphic GN coccobacilli, obligate intracellular parasites, developmental cycle similar to Chlamydia, replicates in phagocytes, form inclusions called morulae). Specific examples: Ehrlichia chaffeensis, Anaplasma phagocytophilum, Diagnostics: peripheral blood smear, Wright, Giemsa stain, Histologic stain, Nucleic amplification testing (PCR), Serology (retrospective)
• Coxiella burnetii - causes Q fever; obligate intracellular GN, Zoonotic (cattle, sheep, goats), found in urine, feces, milk, birth products; remains in soil (spore-like lifecycle), transmitted via inhalation of infectious aerosols, complication: endocarditis, death

Miscellaneous GNR

• Bartonella spp. (e.g., B. quintana, B. henselae) , Spirillum minus, Klebsiella granulomatis, Chryseobacterium, and Chromobacterium violaceum

Test your knowledge on C.trachomatis serovars, their infections, and diagnostic methods. This quiz covers important topics like trachoma, LGV, and other bacterial diseases. Challenge yourself with questions on infection resistance and diagnostic tests.