Microbiology Quiz: Antibiotic Resistance and Chlamydia
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Questions and Answers

What gene is primarily associated with methicillin-resistant Staphylococcus aureus (MRSA)?

  • mecC gene
  • mecB gene
  • mecD gene
  • mecA gene (correct)

Which mechanism describes anaerobes being intrinsically resistant to aminoglycosides?

  • Transformation mechanism
  • Bypass mechanism (correct)
  • Constitutive resistance
  • Inducible resistance

What type of DNA are plasmids classified as?

  • Extrachromosomal circular DNA (correct)
  • Chromosomal DNA
  • Double-stranded RNA
  • Linear DNA

What type of resistance expression is characterized by organisms constantly expressing a resistance mechanism?

<p>Constitutive resistance (D)</p> Signup and view all the answers

Which of the following statements is true regarding Chlamydia species?

<p>They are dependent on hosts for ATP. (B)</p> Signup and view all the answers

What form of Chlamydia is considered infectious?

<p>Elementary body (A)</p> Signup and view all the answers

The major outer membrane protein (MOMP) in Chlamydia is used for:

<p>Species and subspecies identification (C)</p> Signup and view all the answers

Chlamydia trachomatis has how many recognized biovars?

<p>Three biovars (B)</p> Signup and view all the answers

What does β-lactamase testing detect?

<p>Resistance to penicillinase-susceptible penicillins (D)</p> Signup and view all the answers

Which of the following methods is NOT used for β-lactamase testing?

<p>MIC (C)</p> Signup and view all the answers

Which organisms typically produce ESBLs?

<p>Klebsiella spp. (C)</p> Signup and view all the answers

What is the outcome when ESBL-producers are tested against cephalosporins?

<p>They are reported as resistant (D)</p> Signup and view all the answers

What color indicates a positive result in the Nitrocefin test for β-lactamase?

<p>Red (D)</p> Signup and view all the answers

Which of the following statements about inducible β-lactamase expression is true?

<p>It requires exposure to an inducing agent. (A)</p> Signup and view all the answers

Which β-lactamase inhibitor is commonly used to confirm ESBL production?

<p>Clavulanic acid (C)</p> Signup and view all the answers

Which organisms are known for constitutive expression of β-lactamases?

<p>Haemophilus and Neisseria spp. (C)</p> Signup and view all the answers

What type of disease is caused by Bartonella henselae?

<p>Bacillary angiomatosis (B), Cat-scratch disease (C)</p> Signup and view all the answers

Which method is used for the detection of 'Donovan bodies' in Klebsiella granulomatis?

<p>Giemsa-stained tissue smears (B)</p> Signup and view all the answers

What is the transmission method for Spirillum minus?

<p>Rat bites and scratches (D)</p> Signup and view all the answers

Which of the following is a characteristic of Barttonella henselae?

<p>Slow growth on CHOC and BAP (A)</p> Signup and view all the answers

Which staining method is associated with detecting organisms in tissue from Bartonella infections?

<p>Warthin-Starry stain (B)</p> Signup and view all the answers

What is a common characteristic of Ureaplasma urealyticum?

<p>It causes non-chlamydial urethritis. (C)</p> Signup and view all the answers

What is required for specimen collection of Mycoplasma and Ureaplasma?

<p>Immediate plating or storage at -70°C. (A)</p> Signup and view all the answers

What is typically observed in cultures of Mycoplasma?

<p>Fried-egg colony morphology. (A)</p> Signup and view all the answers

How long are cultures typically incubated for M.pneumoniae?

<p>3-4 weeks. (B)</p> Signup and view all the answers

What is the recovery rate sensitivity for Mycoplasma from cultures?

<p>40% (A)</p> Signup and view all the answers

What type of organisms do the Rickettiaceae family primarily consist of?

<p>Obligate intracellular parasites. (D)</p> Signup and view all the answers

Which of the following methods is used for the diagnosis of M.pneumoniae?

<p>Radiography. (A)</p> Signup and view all the answers

What is not a characteristic feature of a culture for Ureaplasma?

<p>Fried-egg colony morphology. (C)</p> Signup and view all the answers

Which type of immune response is primarily associated with Ehrlichia chaffeensis?

<p>Cell-mediated immunity (B)</p> Signup and view all the answers

Which of the following is a characteristic of Anaplasma phagocytophilum?

<p>Infects neutrophils (C)</p> Signup and view all the answers

What is one of the primary symptoms associated with Coxiella burnetii infection?

<p>Flulike symptoms (B)</p> Signup and view all the answers

What kind of organism is Coxiella burnetii classified as?

<p>Obligate intracellular gram-negative coccobacilli (D)</p> Signup and view all the answers

Which diagnostic method is noted for having 90% sensitivity and 100% specificity for Ehrlichia and Anaplasma identification?

<p>Nucleic acid amplification testing (PCR) (B)</p> Signup and view all the answers

Which zoonotic disease is associated with Coxiella burnetii?

<p>Q fever (A)</p> Signup and view all the answers

What type of cells do both Ehrlichia chaffeensis and Anaplasma phagocytophilum replicate within?

<p>Phagocytes (D)</p> Signup and view all the answers

What symptom is commonly associated with Bartonella quintana infection?

<p>Trench fever (B)</p> Signup and view all the answers

What type of infection is associated with psittaci?

<p>Acute lower respiratory infection (B)</p> Signup and view all the answers

Which term describes the lowest concentration of antibiotic that kills more than 99.9% of an organism?

<p>Minimum bactericidal concentration (MBC) (C)</p> Signup and view all the answers

What does 'susceptible' indicate in antimicrobial susceptibility testing?

<p>The organism is inhibited by the standard drug level (B)</p> Signup and view all the answers

What is the purpose of the Clinical and Laboratory Standards Institute (CLSI)?

<p>Create interpretative standards for susceptibility testing methods (A)</p> Signup and view all the answers

What is an important factor in selecting an ideal antibiotic?

<p>It has selective toxicity without harming the patient (D)</p> Signup and view all the answers

In susceptibility testing, which group reports the primary antimicrobial agents used first?

<p>Group A (D)</p> Signup and view all the answers

What does it mean if an organism is categorized as 'intermediate'?

<p>Higher doses may be needed for inhibition (D)</p> Signup and view all the answers

What is the primary requirement for preparing an inoculum for antimicrobial susceptibility testing?

<p>Ensuring the culture is pure (A)</p> Signup and view all the answers

Flashcards

What is MRSA?

A type of Staphylococcus aureus that is resistant to the antibiotic methicillin. It carries the mecA gene, which encodes for a low-affinity penicillin-binding protein (PBP). This PBP is less effective at binding to penicillin, making the bacteria resistant.

What is VRSA?

A type of Staphylococcus aureus that is resistant to the antibiotic vancomycin. It is considered highly dangerous due to the limited treatment options available.

What is the mecA gene?

A gene found in certain bacteria, including MRSA. It encodes for a penicillin-binding protein (PBP) with a lower affinity for penicillin, making the bacteria resistant to this antibiotic.

What are bypass mechanisms in antibiotic resistance?

A mechanism of antimicrobial resistance where the bacteria bypasses the normal target of the antibiotic. This can happen due to various factors like changes in the bacterium's metabolic pathways or the development of alternative targets for essential processes.

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How is antimicrobial resistance disseminated?

The transfer of genetic material between bacteria, allowing for the spread of antibiotic resistance genes. This can occur through transformation, transduction, or conjugation.

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What is constitutive resistance?

A type of bacterial resistance where the bacteria constantly expresses the resistance mechanism, even in the absence of the antibiotic.

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What is inducible resistance?

A type of bacterial resistance where the resistance mechanism is only expressed when exposed to the antibiotic.

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What is heterogeneous resistance?

A type of bacterial resistance where some bacteria within a population express the resistance mechanism, while others do not.

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Minimum Inhibitory Concentration (MIC)

The lowest concentration of an antibiotic that prevents visible growth of a specific bacteria.

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Minimum Bactericidal Concentration (MBC)

The lowest concentration of an antibiotic that kills at least 99.9% of a specific bacteria.

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Susceptible (S)

A category in susceptibility testing indicating that an organism is likely to be inhibited by the recommended dose of a specific antibiotic.

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Intermediate (I)

A category in susceptibility testing indicating that an organism may need a higher dose or longer treatment duration of the antibiotic to be effectively inhibited.

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Resistant (R)

A category in susceptibility testing indicating that an organism is unlikely to be inhibited by the recommended dose of a specific antibiotic, even at achievable levels in the patient.

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Clinical and Laboratory Standards Institute (CLSI)

A standardized set of antibiotic susceptibility testing methods, including guidelines for interpretation of results.

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Test Battery

A collection of 10-15 antibiotic agents used to test the susceptibility of commonly isolated bacteria.

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Preparation of Inoculum

A crucial step in antibiotic susceptibility testing, requiring a pure culture of bacteria to be grown in a broth medium to determine the bacteria's concentration per milliliter.

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What are beta-lactamases?

A group of enzymes produced by bacteria that break down the beta-lactam ring of penicillin and cephalosporin antibiotics, leading to antibiotic resistance.

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What is constitutive beta-lactamase production?

A type of beta-lactamase that is produced by bacteria even in the absence of the antibiotic.

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What is inducible beta-lactamase production?

A type of beta-lactamase production where bacteria only produce the enzyme in the presence of a specific antibiotic.

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What are extended-spectrum beta-lactamases (ESBLs)?

A group of beta-lactamases that are frequently found in Klebsiella, Proteus, and E. coli. They inactivate many cephalosporins and monobactams.

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What are carbapenemases?

A type of beta-lactamase that is particularly problematic as it can break down carbapenems, a class of antibiotics that are often used for serious infections.

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What is the nitrocefin test?

A test that uses the chromogenic cephalosporin nitrocefin to detect the presence of beta-lactamases. If the enzyme breaks down nitrocefin, a color change occurs.

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What is the acidimetric beta-lactamase test?

A test used to detect beta-lactamases based on the production of penicilloic acid resulting from breakdown of penicillin. It involves a pH indicator.

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What is the iodometric beta-lactamase test?

A test that uses starch-iodine to detect beta-lactamase activity by monitoring the breakdown of penicillin and its effect on the indicator.

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Bartonella

A genus of bacteria that includes Bartonella henselae, known for causing cat-scratch disease, and Bartonella quintana, responsible for trench fever.

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Cat-scratch disease

A disease caused by Bartonella henselae, typically transmitted by cat scratches or bites. Symptoms include localized lesions and fever, usually resolving within 2-4 months.

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Bacillary angiomatosis (BA)

A rare but serious infection caused by Bartonella henselae. It is characterized by the proliferation of blood vessels, forming a mass-like structure, often seen in individuals with weakened immune systems (e.g., HIV patients).

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Spirillum minus

A small, gram-negative rod-shaped bacterium with helical coils. It is the causative agent of rat-bite fever (sodoku), a disease transmitted through bites or scratches from infected rats.

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Klebsiella granulomatis

A gram-negative rod-shaped bacterium that causes granuloma inguinale, also known as donovanosis. This sexually transmitted infection (STI) is characterized by ulcerative lesions in the genital area.

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Ureaplasma urealyticum

These are bacteria that infect the urinary tract by adhering to the urethra. Infections are more common in men and associated with inflammation of the urethra.

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Mycoplasma & Ureaplasma

These are bacteria that can be transmitted to newborns during delivery. They may cause meningitis, making it important to recognize them in a newborn with a negative gram stain.

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Mycoplasma and Ureaplasma cultures

These bacteria require special media for growth. They grow slowly and have distinctive colony morphologies.

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Mycoplasma Identification

These bacteria can be identified through various methods. The most common is the detection of IgG and IgM antibodies, which indicate exposure to the organism. Other helpful tests include complement fixation and enzyme immunoassay.

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Rickettsia and Ehrlichia

They infect via arthropod vectors and reside inside cells, meaning they require a host organism to survive.

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Mycoplasma

They are a diverse group of bacteria often associated with pneumonia. They are difficult to grow, requiring special media like A7, E agar, and U broth, enriched with sterols and penicillin. They take up to 4 weeks to grow and have distinctive colony morphologies.

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Ureaplasma

This is a type of bacteria that can be cultured on special media like SP4 Agar. They produce small colonies and can be visualized under a microscope. Unlike mycoplasma, they colonize the urogenital tract.

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Mycoplasma and Ureaplasma specimen collection

Proper specimen collection and transport are crucial for the accurate identification of these organisms since they are sensitive to drying and heat. They are usually inoculated at the bedside and require specific transport media like tryptic soy agar with penicillin or SP4 (sucrose phosphate buffer, serum) to ensure their viability.

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Rickettsiae and their Serogroups

A group of bacteria that are closely related to rickettsiae but can be differentiated by their agglutination reactions with specific antibodies. They are associated with various diseases like spotted fever and typhus.

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Immunohistochemical Staining

A technique used to identify the presence of specific antigens in tissue samples. Antibodies are attached to an enzyme, which reacts with the antigen, producing a color change in the sample. This technique is helpful for diagnosing infections with Ehrlichia and Anaplasma.

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Ehrlichia and Anaplasma

A type of bacteria that primarily infects the blood cells of mammals. They are obligate intracellular parasites, similar to Chlamydia, and have a distinct developmental cycle.

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Morulae

Characteristic structures formed inside infected cells by Ehrlichia and Anaplasma. These structures are clusters of bacteria in a morula-like formation.

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Ehrlichia chaffeensis

A type of ehrlichia that causes human monocytic ehrlichiosis (HME). It is transmitted by tick bites and occurs mainly in the Southern US, causing fever, headache, muscle pain, and other symptoms.

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Anaplasma phagocytophilum

A type of ehrlichia that causes human granulocytic ehrlichiosis (HGE), transmitted by tick bites, typically found in the Northern US. It infects neutrophils and exhibits similar symptoms as Ehrlichia chaffeensis.

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Coxiella burnetii

A type of bacteria responsible for Q fever, a zoonotic disease often transmitted through inhalation of aerosols contaminated with animal products. Coxiella burnetii is an obligate intracellular parasite and can remain viable in the environment for long periods.

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Miscellaneous GNR

A group of diverse bacteria that includes several significant pathogens. Examples include Bartonella quintana, responsible for trench fever, and Klebsiella granulomatis, which causes granuloma inguinale.

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Study Notes

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 (includes antimicrobial and anticancer drugs)
  • Bactericidal - "cide" means kill (agent kills bacteria)
  • Bacteriostatic - "static" means no change (agent inhibits bacteria)
  • Spectrum of activity
    • Narrow spectrum - covers only certain groups
    • Broad spectrum - covers gram-positive and gram-negative
  • Mechanism of action - the way an agent harms an organism
    • 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 the sum of the activity of individual antimicrobial agents (1+2=3)
  • Synergism - effect is amplified (1+2=4)
  • Antagonism - one agent interferes with the activity of another (1+2=1)

Cell Wall Characteristics

  • Not found in mammalian cells
  • Cell wall characteristics affect the spectrum of antibiotic activity
  • Gram-positive - thick peptidoglycan cell wall
  • Gram-negative - thin peptidoglycan surrounded by an outer membrane
  • Peptidoglycan is also known as the murein layer

Gram-Positive and Gram-Negative Cell Walls

  • Gram-positive cell wall structure: Shows a thick layer of peptidoglycan, teichoic acid, and an inner cytoplasmic membrane
  • Gram-negative cell wall structure: Shows a thin peptidoglycan layer and an outer membrane with lipopolysaccharides (LPS), proteins, lipoproteins etc, and an inner cytoplasmic membrane

Gram-Positive and Gram-Negative Cell Walls - Inner (Cytoplasmic) Membrane

  • 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

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
    • 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
  • DNA gyrase
  • DNA transcription/RNA polymerase

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
  • Tetracyclines
    • Doxycycline, minocycline, tetracycline
    • Inhibit 30S ribosomal subunit
    • Broad spectrum, including mycoplasma and chlamydia
  • Macrolides
    • Erythromycin, clarithromycin, azithromycin
    • Inhibit 50S ribosomal subunit
    • Broad spectrum
  • Lincosamides (clindamycin) and Streptogramins (dalfopristin/quinupristin)
    • Inhibit 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.

Mechanisms of Resistance

  • Intrinsic
    • Resistance resulting from normal genetic, structural, or physiologic state of the microorganism.
    • Naturally occurring
  • Acquired
    • Resistance resulting from alterations in cellular physiology or structure due to genetic change.
    • Transfer or mutation to genetic code
  • Decreased uptake or accumulation
    • Impermeability
    • Efflux
    • Biofilms
  • Enzymatic inactivation or modification
    • Target site modification
    • Acquisition of new target
    • Pathway bypass

Mechanisms of Resistance (specific features)

  • Impermeability of cell wall
    • LPS and porins restrict entry of antimicrobials
    • All GN resistant to vancomycin
    • Cannot cross outer membrane
  • 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 GP and GN organisms
    • Most common = penicillinase
    • Virtually all GN bacteria are intrinsically resistant via beta-lactamase production, Staph are common GP producers.
    • Cephalosporins were created to be forever resistant to all beta-lactamase enzymes
    • Extended-spectrum beta-lactamases (ESBLS) - inactivate all penicillins and cephalosporins
  • Target site modification
    • Drug binds poorly (or not at all) to target site, altering of PBP decreases affinity
    • Mutations to topoisomerase IV and DNA gyrase increases resistance
    • Addition of methylase to ribosome decreases binding

Mechanisms of Resistance (Continued)

  • 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)
  • Acquisition of new target site
    • mecA gene in MRSA encodes for a low affinity PBP, resistant to all penicillins, found in mobile SCCmec cassette
  • Bypass mechanisms
    • Organisms circumvent consequences of antimicrobial action
    • Anaerobes are intrinsically resistant to aminoglycosides (lack oxidative electron transport system)

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 expresses resistance
  • Heterogeneous - some bacteria in population express resistance (MRSA)

Chlamydia

  • Three species cause human diseases: C. trachomatis, C. pneumoniae, C. 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 and multiply only inside animal or host epithelial cells
  • Replication cycle
    • Elementary body (EB) - infectious
    • Reticulate body (RB) - non-infectious
    • EB infects host cell, organizes into RB, then multiply
    • Host cell ruptures and releases EB
  • Antigens
    • Outer membrane similar to Gram-negative but no peptidoglycan
    • Components = Lipopolysaccharide (LPS), Major outer membrane protein (MOMP)
    • Species subdivided into serotypes (serovars) based on MOMP

C. trachomatis biovars

  • Trachoma (serovars A-K) - chronic eye infection
  • Inclusion Conjuctivitis (serovars D-K) - milder eye infections, colonizes nasopharynx
  • Lymphogranuloma venereum (LGV, serovars L1, L2, L2a, L2b, L3) - invasive urogenital disease
  • Newborn Chlamydia trachomatis - passed through birth canal, conjunctivitis, pneumonia, or nasopharyngeal infection

C. Pneumoniae

  • Mild or asymptomatic infection in young adults
  • Prolonged pharyngitis, bronchitis, or pneumonia
  • Potential risk factor for Guillain-Barré Syndrome, asthma, and cardiovascular disease

C. psittaci

  • ornithosis (psittacosis, parrot fever)
  • zoonotic infection
  • occupational hazard for pet bird handlers and poultry workers
  • acute lower respiratory infection, cultures are not sensitive; diagnosed serologically

Antimicrobial Susceptibility Testing and Therapy

Ideal Antibiotic

  • Selective toxicity - exerts effects on the agent of disease without harming the patient
  • Treats diseases caused by both Gram-positive and Gram-negative organisms
  • Host does not develop hypersensitivity or allergic reaction
  • Agent penetrates into tissues and crosses the blood-brain barrier
  • Cost is minimal

Susceptibility Terms

  • Minimum inhibitory concentration (MIC): lowest antibiotic concentration that inhibits microbial growth
  • Minimum bactericidal concentration (MBC): lowest antibiotic concentration that kills >99.9% of microorganisms
  • Susceptible/sensitive: interpretive category; indicates organism is inhibited by a particular dose of antibiotic.
  • Intermediate: interpretive category; indicates organism may require higher dose of antibiotic for longer period to be inhibited
  • Resistant: interpretive category; indicates organism is not inhibited by recommended antibiotic dose or achievable level

Susceptibility Standards (e.g., CLSI or NCCLS)

  • Develops standards for susceptibility methods
  • Testing categories: susceptible (S), intermediate (I), resistant (R)

Test Batteries

  • Panel of 10-15 antimicrobial agents for routine testing
  • Panels selected based on organism group (GN, GP, urine)
  • Group categorization for primary agents and any supplemental groups

Preparation of Inoculum

  • Requires pure culture
  • Most important step of all susceptibility testing
  • Determine # of bacteria per mL
  • Compare to McFarland standards (e.g., McFarland 0.5)

Standardized Testing Methods

  • Broth dilution method: antibiotic suspended in a solution
  • Agar dilution method: antibiotic incorporated into the agar medium
  • Diffusion method: uses antibiotic-impregnated disks or strips
  • Special methods: additional techniques for specific organisms/ situations

Broth Dilution Tests

  • Provides MIC
  • Uses Mueller-Hinton broth for serial dilutions, adding 2-5% horse blood (when needed).
  • Final bacterial concentration = 5 x 105 CFU/mL.
  • Methods: macrodilution (tubes with 1-2 mL broth), microdilution (microtiter trays with 0.05-0.1 mL)
  • Internal controls (growth control, sterility check)
  • MBC involves culturing on BAP to get bacterial colonies vs. initial colony count.

Serum Bactericidal Test

  • Tests patient's serum (with antibiotics) against infecting organism
  • Incubation overnight and examining for growth
  • Measures peak and trough serum samples

Microdilution

  • Similar to macrodilution, but uses plastic panels with wells for precise concentrations.

Agar Dilution Tests

  • Semi-quantitative, incorporated antimicrobial agents into agar medium, uses agar plates with varying concentrations, standardized suspension is inoculated and incubated and examined for growth.

Disk Diffusion Tests (Kirby-Bauer)

  • Paper disks impregnated with antibiotic placed on inoculated plates.
  • Incubated at 35˚C → measure zone of inhibition.
  • Larger zones = more antimicrobial activity/greater diffusion.
  • No zone = complete resistance.
  • Mueller-Hinton Agar criteria = standard media, QC strains are tested with E. coli, S. aureus & P. aeruginosa
  • Agar depth: 4 mm, too thin = increases zones, too thick = decreases zones, False susceptibility + resistance
  • pH 7.2-7.4 (important for results) , Decreased pH = decreased activity of aminoglycosides, erythromycin, and clindamycin & increased activity of tetracyclines, Increased pH has opposite effect.
  • Fixed cation concentration (Ca++ & Mg++) = increased concentrations = decreased aminoglycosides activity against P. aeruginosa and decreased activity of tetracyclines

Diffusion E-Test

  • Agar plates inoculated → plastic strip containing gradient of antimicrobial agent on agar surface → incubated & examined for an elliptical zone of inhibition.

Test Performance

  • Inoculum from overnight growth
  • MHA plus sheep blood for strep
  • Incubated in 5-7% CO2
  • Use Chocolate MHA as needed
  • Store disks properly
  • Check for potency
  • Allow disks to warm to room temperature before use

Reading and Interpretation

  • Lawn of growth must be confluent
  • Ignore small colonies, Proteus Spp., swarming
  • Contamination & mixed cultures cannot be ignored
  • Resistant subpopulation

Special Tests

  • Beta-lactamases, ESBLs, carbapenemases, altered PBPs
  • Inducible macrolide resistance
  • Oxacillin resistance
  • High-level aminoglycoside resistance
  • Anaerobes

Beta-Lactamase Tests

  • Detects beta-lactamase-mediated resistance (penicillin, ampicillin)
  • Rapid tests are useful for H. influenzae, N. gonorrhoeae, Morexella catarrhalis → Staphylococcus spp.,Bacteroides spp.
  • Methods
    • Nitrocefin
    • Acidimetric
    • lodometric

ESBLs (Extended-Spectrum Beta-Lactamase) Tests

  • Typically seen in Klebsiella, Proteus, & E. coli
  • Test against cephalosporins & monobactams (cefpodoxime, ceftazidime, cefotaxime, ceftriaxone, aztreonam)
  • Confirm with Clavulanic acid

Carbapenemases

  • Initially identified in K. pneumoniae (KPC)
  • Now in many Enterobacteriaceae
  • Resistance to carbapenems (imipenem, meropenem, ertapenem)

Altered PBPs

  • Detect penicillin resistance in pneumococci w/ Oxacillin disk → more accurate than penicillin disk

Macrolide Resistance

  • Isolate appears erythromycin resistant but clindamycin is susceptible
  • Inducible clindamycin resistance may be present
  • D test must be performed

Oxacillin-Resistant Staphylococci (MRSA)

  • Oxacillin is more reliable than methicillin
  • Belong to same class of agents as penicillinase-resistance penicillins
  • Isolate resistant to one = resistant to all
  • Mec-A-mediated resistance makes staph heteroresistant/difficult to detect
  • Modifications
    • Supplement MH with 2% NaCl
    • Incubate at <35°C for 24 hrs

Vancomycin Resistance

  • MRSA isolates with resistance to vancomycin (VISA, VRSA)
  • Recommended method of detection in staph/enterococci — Broth dilution
  • Vancomycin agar screen (at 6 µg/mL) for heteroresistant strains (hVISA)

High-Level Aminoglycoside Resistance in Enterococci

  • Enterococci may be treated with ampicillin/penicillin
  • Bacteriostatic only
  • Combined with aminoglycoside to achieve bactericidal effect
  • Enterococci are intrinsically resistant
  • Synergistic with cell wall active agent High-level aminoglycoside resistance due to enzymatic inactivation. Detect using broth, agar, or disk. Screening concentrations (broth = 500 to 1000 µg/mL)

Anaerobes

  • Agar dilution (use supplemented Brucella laked sheep blood agar)
  • Broth microdilution (Brucella broth with lysed horse blood)
  • Inoculum is higher (1 x 106 CFU/mL)
  • Incubation is longer (48 hrs)

Automated Systems

  • Optical methods to detect susceptibility endpoints
  • Turbidometric
  • Hydrolysis of fluorometric growth substrate (analyses in shorter period: 5-15 hours)
  • Use microprocessors

QC (Quality Control)

  • Interpretations determined by CLSI
  • Reference strains (ATCC)
  • Defined susceptibility/resistance patterns
  • Range of endpoints (S, I, R)
  • Endpoint is on-scale
  • Acceptable results defined → Weekly testing
  • Supplemental QC: periodic testing (MRSA, ampicillin-resistant E. cloacae)
  • Predictor Drugs
    • Staphylococci resistant to oxacillin
    • Enterococci with high-level aminoglycoside resistance
    • Enterococci resistant to ampicillin

Susceptibility Testing (required for specific organisms)

  • Full susceptibility testing is required for staphylococci, S. pneumoniae, viridans strep (if from a sterile site), enterococci, Enterobacteriaceae, P. aeruginosa, & Acinetobacter spp
  • Testing occasionally required (beta-lactamase testing) for H. influenzae, N. gonorrhoeae, Morexella catarrhalis, anaerobes, Beta-hemolytic streptococci, Neisseria meningitidis, Listeria monocytogenes

Miscellaneous Bacteria

  • Mycoplasma, Ureaplasma, Rickettsia, Ehrlichia, Coxiella burnetii, miscellaneous GNR.

Mycoplasma & Ureaplasma

  • Belong to class Mollicutes
  • Size of a large virus; smallest self-replicating microorganisms
  • Colonize mucous membranes of RT & UGT
  • Humans and animals
  • Sexual contact
  • Mother-to-child and respiratory secretions
  • No cell wall, do not gram stain, pleomorphic
  • Utilize sterols to create membrane support; resistant to penicillin & cephalosporins
  • Slow-growing, fastidious; cell culture.
  • Mycoplasma pneumoniae (primary atypical pneumonia; walking pneumonia)
  • Mycoplasma hominis (colonizes UGT, opportunistic pathogen — PID, salpingitis, pyelonephritis)
  • Other mycoplasma species (M. genitalium, M. fermentans, and M. penetrans)

Ureaplasma urealyticum

  • Colonizes urogenital tract, nonchlamydial & nongonococcal urethritis in men, associated with upper genital tract infections in women
  • Hydrolyzes urea.
  • Can be transmitted to newborns at delivery; set up culture if meningitis is suspected; negative gram stain & culture.

Rickettsiae & Similar Organisms

  • Arthropod-borne
  • Obligate intracellular parasite
  • Gram-negative coccobacilli
  • Rickettsia spp.
  • Ehrlichia spp.
  • Coxiella burnetii
  • Predilection for endothelium of blood vessels; never grown in cell-free media
  • Nonmotile
  • Two groups (spotted fever group, typhus group)

Rickettsial Spotted Fever Group

  • R. rickettsiae: Rocky Mountain Spotted Fever, vector is ticks, flu-like symptoms, characteristic rash on hands & feet, disseminated infection (lungs, brain, heart), 20% mortality rate if untreated
  • R. akari: Rickettsialpox, vector is mouse mites, similar to RMSF but milder, rash on face, body, and extremities (not on palms & soles), Eastern US

Rickettsial Typhus Group

  • R. typhi: Endemic typhus or murine typhus, vector is rat fleas, flu-like symptoms & +/- rash
  • R. prowazekii: Epidemic louse-borne typhus, war & natural disasters, vector is human body & squirrel louse, flu-like symptoms, rash on palms, soles, face; 40% mortality if untreated, Brill-Zinsser disease = reactivation of infection.

Orientia tsutsugamushi

  • Formerly Rickettsia, causes scrub typhus, vector is rat chigger, flu-like symptoms & rash.

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Test your knowledge on antibiotic resistance mechanisms and Chlamydia species with this comprehensive quiz. Explore questions related to MRSA, β-lactamase testing, and more. Perfect for microbiology students looking to reinforce their understanding of these critical topics.

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