Microbiologists must be able to differentiate potential pathogens from the
usual flora
What is an important component of quality control that is accomplished by recognizing organisms by their colonial characteristics?
the ability of the microbiologist to determine whether the inoculum is mixed and to ascertain whether the results generated by a commercial or automated system correlate with the suspected identification of the organism
How long are plates observed for?
18 to 24 hours post-culture
The interpretation of primary cultures, commonly referred to as plate reading, is...
actually a comparative examination of the colony morphology of microorganisms growing on various culture media
MacConkey's agar (MAC):
gram-negative organisms, particularly enterics.- will use differential medias : for Lactose fermenters(Pink) : and Non-lactose fermenters
Enteric pathogens will produce what color colonies on a mac plate?
clear/colorless
Escherichia coli/Citrobacter:
dry pink colonies- produces, pink colonies with a surrounding halo of pink precipitated bile salts
Klebsiella/Enterobacter-like organisms:
large mucoid pink colonies- produces large, mucoid pink colonies that occasionally have cream-colored centers
Comparative analysis of the culture media is very important in initial interpretation because:
- Microorganisms grow on culture media in the same proportion or concentration in which they are present in the clinical specimen
What are Alpha Hemolysis?
partial clearing of blood that results in a green discoloration of the medium; Streptococcus pneumoniae, certain viridans strep
What are Beta Hemolysis?
complete clearing of blood cells around or under the colonies because of the complete lysis of RBC's; S. pyogenes; S. agalactiae; Listeria monocytogenes
Non-hemolytic colonies are sometimes referred to as:
Delta hemolytic
How are the colony characteristics determined?
- Hemolysis- Size- Form/Margin: Edge- Elevation- Density- Color- Consistency- Pigment- Odor
How are Colony size described as?
- Large- Medium- Small - Pinpoint
How are the edge of the colonies described as?
- Smooth- Filamentous- Round/rhizoid- Irregular
Swarming colonies are described as?
hazy blanket of growth on surface that extend well beyond the streak lines; Proteus spp.
Feet and stars of colonies are described with what?
Yeast
Elevation of colonies are described as?
- Raised- Convex- Flat- Umbilicate- Umbonate
Density of colonies are described as?
- Transparent- Translucent- Opaque
Colors of colonies may be?
- White- Gray- Yellow- Buff(pale yellow brown)
What type of odor would you smell with P.aeruginosa?
Fruity and grapelike odor
The identification of bacteria has been based on what?
- Colony morphology - Gram staining- Biochemical testing; Phenotype of microorganisms
Serotyping:
Uses antibodies to detect expressed proteins or specific antigens on the surface of a bacterium
Molecular biology is based on:
Genotypic characteristics of the organism; Nucleic acid assays
Different types of sugars used by bacteria:
- Lactose- Maltose- Rhamnose- Sucrose- Raffinose- Arabinose
Oxidation:
Utilize carbohydrates aerobically
Fermentation:
Utilize carbohydrates anaerobically
Asaccharolytic:
Do not utilize carbohydrates; instead they use other organic molecules such as amino acids, for energy & carbon sources
Fermentation process:
glucose enters the glycolysis pathway, resulting in the formation of pyruvic acid
Oxidative/Fermentative:
Acid production (A) is indicated by the color indicator changing to yellow
Non Oxidative/Fermentative:
in an Alkaline environment the color indicator is blue
Alkaline slant/alkaline butt:
(K/K); No fermentation
Alkaline slant/acid butt:
(K/A); Glucose fermentation only
Acid slant/acid butt:
(A/A); Lactose, sucrose fermentation or both
H2S production:
Black precipitate
Gas
Bubbles or splitting of media
Ortho-Nitrophenyl-B-D-Galactopyranoside (ONPG) Test:
test that distinguishes delayed lactose fermenters from non-lactose fermenters of Enterobacteriaceae
What test indicates a acetoin production?
Voges-ProskauerPositive; Red color
What test is used to measure the enzymatic ability of an organism to decarboxylate an amino acid to form an amine?
Decarboxylase/Dihydrolase tests; Positive: Alkaline(Purple) color change; Negative: No color change
Citrate Utilization:
used to identify organisms capable of using sodium citrate as the sole carbon source and inorganic ammonium salts as the sole nitrogen source; Principle: - Bacteria that can grow on this medium produce an enzyme, citrate-permease, capable of converting citrate to pyruvate
What are the expected results of a citrate test?
Positive: Growth on medium, with or without a change in colorNegative: Absence of growth
Indole Production:
Test used to identify organism that produce the enzyme Tryptophanase
What are the expected results of a indole test?
Positive: Pink to Wine colored ringNegative: No color change
What is the principle of Motility?
The inoculum is stabbed into the center of a semisolid agar deep; It is evident by a diffuse zone of growth extending out from the line of inoculation
What are the expected results of a motility test?
Positive: will spread out into the medium from the site of inoculation. A hazy appearance throughout the motility media or movement away from a stabNegative: Non-motile organism remain at the site of inoculation
Oxidase:
this test determines the presence of cytochrome oxidase activity in microorganisms for the ID of oxidase-negative Enterobacteriacease; Dark purple positive results within 10 seconds
Lysine Iron Agar (LIA):
contains Lysine- Peptones- Small amount of glucose- Ferric ammonium citrate- Sodium thiosulfate
What is the bacterial enzyme for PYR?
Pyrrolidonyl Arylamidase
What are the 5 Antibiotic Targets and Mechanisms of Action
- Inhibition of Bacterial Cell Wall Biosynthesis- Inhibition of Folate Synthesis- Interference with DNA Replication- Interference with DNA Transcription- Interference with mRNA Translation
In gram-positive bacteria, the peptidoglycan later is substantially ______ and more multilayered than in gram-negative bacteria.
thicker
The outer membrane of gram-negative bacteria is composed of
- Lipopolysaccharides- Phospholipids- Porin proteins
B-Lactams Drugs:
- Penicillins(penicillin, ampicillin, oxacillin)- Cephalosporins(cefazolin, cefoxitin, ceftriaxone, cefepime)- Carbapenems(imipenem)- Monobactams- Penems
What are the extended spectrum B-Lactamases drugs?
- Penecillins- Cephalsporins- Monobactams
Which drug class targets the penicillin-binding proteins (PBP)?
B-Lactams
What is the four-membrane B-lactam ring?
the active moiety of B-lactam
Glycopeptides and Glycolipopeptides Drugs:
- Vancomycin- Teicoplanin
Which drug class targets peptidoglucan units?
Glycopeptides and Glycolipopeptides
Lipopeptides drugs:
- Daptomycin- Polymixin B
Which drug class targets the cell membrane?
Lipopeptides
Which drug classes affect the cell wall biosynthesis?
- B Lactams- Glycopeptides & Glycolipopeptides - Lipopeptides
Trimethoprim-sulfamethoxazole drug:
- Co-trimoxazole
Which drug class target the Folate synthesis?
Trimethoprim-sulfamethoxazole; targets tetrahydrofolic acid synthesis inhibitors
Which drug class affects DNA Replication?
Fluroquinolones; Targets Topoisomerase 2 & 4
Fluroquinolones drugs:
- Ciprofloxacin- Gemifloxacin
Which drug class affects DNA Transcription?
Rifamycins; Targets DNA-dependent RNA polymerase
Rifamycins drugs:
- Rifamycins- Rifampin- Rifapentine
Interference with mRNA Translation Drug Classes:
- Aminoglycosides- Tetracyclines- Macrolides- Streptogramins- Phenicols
Which class of drugs targets the 30S ribosome?
- Aminoglycosides- Tetracyclines
Which drug class are of polyketide class?
- Tetracycline- Doxycycline- Minocycline
Which class of drugs targets the 50S ribosome?
- Macrolides- Streptogramins- Phenicols- Lincosamides- Oxazolidinones
Aminoglycosides drugs:
- Gentamicin- Tobramycin- Streptomycin- Kanamycin
Macrolides drugs:
- Erythromycin- Azithromycin- Clarithromycin
Streptogramins drugs:
- Pristinamycin- Dalfopristin- Quinupristin
Phenicols drug:
- Chloramphenicol
Intrinsic Mechanisms of Resistance:
- Impermeability- Biofilms- Efflux- Enzymatic Inactivation
What is a public health issue uniting scientists from across the world to develop strategies to address it?
Antibiotic resistance; Intrinsic; Acquired
Acquired Mechanisms of Resistance:
Exogenous DNA (plasmid, conjugation, transposons, bacteriophase)- Efflux pumps- Target site modification- Acquisition of new targets- Enzymatic inactivation of antibiotics
B-lactamase inhibitors:
structural analogues of the B-lactam antibiotics and function as substrates for B-lactamase, thus reducing their detrimental effects on the B-lactam antibiotic
What uses efflux as an effective mechanism for acquired resistance to macrolides?
Streptococcus pyogenes and Streptococcus pneumoniae
In clinical laboratories, susceptibility testing is usually performed by a
disk diffusion or dilution(Minimal inhibitory concentration) method.; Methods of these are published and frequently updated by the Clinical Laboratory Standards Institute (CLSI)
The inhibition zone size and minimal inhibitory concentration interpretive criteria are established by careful analysis of all of the following components:
- Microbiologic data- Pharmacokinetic data- Clinical studies results before FDA approval
Reasons and Indications for Antimicrobial Susceptibility:
- Probable cause to suspect isolate is involved in patient infection- Susceptibility against particular antimicrobials is uncertain; Not on bacteria known to be susceptible- Epidemiological cut-off values (ECV); Helps identify emerging resistance
Factors to Consider in Susceptibility Testing:
- Host Status- Presence of Other Bacteria and Quality Specimen - Body site from which the organism was isolated
What is one of the most critical steps in susceptibility testing?
Inoculum preparation
Minimal Inhibitory Concentration (MIC):
Lowest concentration of antimicrobial agent required to inhibit the growth of a bacterial isolate
How is the dilution method interpreted? Hint: 3 MIC results values
- Susceptible- Intermediate- Resistant
Susceptible:
interpreted if the patient's infecting organism responds to therapy with an antimicrobial agent using the dosage normally recommended for that type of infection and that species
Resistant:
interpreted as it is unlikely to be inhibited by the usually achievable concentrations of the antimicrobial agent based on the dosages normally used with that drug
Kirby Bauer test:
Swab a 0.5 McFarland standard over the entire surface of an agar plate (Mueller-Hinton)- Add paper disk containing antibiotic concentrations- Used since 1966
E-Test:
Use of an antibiotic strip- uses thin plastic test strips impregnated on the undersurface with an antimicrobial concentration gradient
Production of B-lactamse is a significant mechanism contributing to resistance to some B-lactams in certain organisms such as:
- H. influenzae- N. gonorrhoeae- M. catarrhalis- Staphylococcus spp.- Bacteroides spp.
Modified Methods for Streptococci requires what?
- 2% to 5% horse blood- 5% sheep's blood
Automated systems: Most current instruments use the principle of __________ in a broth medium using a photometer to examine the test wells.
Turbidimetric detection of bacterial growth
What is the most widely used supplemental QC measure to verify results generated on patient isolates?
the use of antibiograms
Antibiogram:
the overall antimicrobial susceptibility profile of a bacterial isolate to a battery of antimicrobial agents
When atypical antibiograms are seen, the results must be verified by:
- Reexamining the disk diffusion plate to ensure that results were properly interpreted- Checking earlier reports to see whether the particular patient previously had an isolate- Repeating the test, if necessary