Test 2: Antibiotics Flashcards
1
Q
Antibiotics
A
Substance produced by a
microorganism that inhibits or kills other microorganisms
2
Q
Antimicrobial agents
A
Chemical substance
produced either by a microorganism or
synthetically that can kill or suppress
microorganisms.
3
Q
Antimicrobial agents may be described by what
A
*Bacteriostatic – inhibits bacterial growth
without killing
*e.g. tetracycline, erythromycin, Trimethoprim
*Bactericidal – kills bacteria
*e.g., aminoglycosides, beta-lactams,
vancomycin
4
Q
What are types of Bacteriostatic drugs
A
Tetracycline, Erythromycin, and Trimethoprim
5
Q
What are types of Bactericidal drugs
A
Aminoglycosides, Beta-Lactams, Vancomycin
6
Q
Antimicrobial agents requirements
A
For an antibiotic to affect the growth
of a microbial cell it must
(i) enter the cell and reach the site of action,
(ii) bind to a target molecule involved in an
essential cell process,
(iii) markedly inhibit this process.
7
Q
Antimicrobial resistance
A
is the ability of
a microorganism to withstand that
agent
8
Q
Antimicrobial susceptibility
A
means that an
agent can kill (or inhibit growth of) the
organism at a specific concentration.
This is the MIC
9
Q
activity of the microbial agent is
A
The degree and mode of
action of an antimicrobial agent
10
Q
Spectrum of activity
A
its effective
against a large variety of organisms–
has broad spectrum
* Active against both gram-positive &
gram-negative; also, unidentified
pathogen
* Disadvantage: kills normal flora
11
Q
Minimum Inhibitory concentration MIC
A
of an antibacterial agent is defined as the
maximum dilution of the product that
will still inhibit the growth of a test
microorganism.
12
Q
Minimal bactericidal concentration
A
is defined as greater than or equal
to 99.9% reduction of visible colony
forming units of a bacterial or fungal
suspension.
13
Q
MIC=MBC when
A
The concentration where there is no growth after incubation
14
Q
how do you treat the person when MIC=MBC
A
Give antibiotics in high amounts and give for a longer period of time
15
Q
MIC= what test tube
A
The high dilution without growth
16
Q
Breakpoint
A
Concentration of
an antibiotic which defines in vitro whether a
species or group of species is susceptible or
resistant to an antibiotic/antimicrobial.
17
Q
Breakpoint separates or defines
A
The interpretive category ( S,I, or R)
18
Q
In breakpoint take in to account
A
wild type distribution of MICs
(wild type-those that do not have acquired
resistance or selected resistance mechanisms)
19
Q
Other important implementations of Breakpoint
A
- Pharmacokinetics/pharmacodynamics
- Clinical outcome studies
20
Q
Cell wall inhibitors
A
- Beta-lactams: contain beta-lactam ring
1. Penicillins
2. Cephalosporins
3. Carbapenems - Fosfomycin: inhibits enzyme at first step of
cell wall synthesis - Glycopeptides/lipoglycopeptides
1. Vancomycin
2. Teicoplanin - Monobactam
1. Aztreonam
21
Q
Types of Beta-Lactams
A
Penicillins, Cephalosporins, and Carbapenems
22
Q
Beta-lactams contain a
A
Beta-lactam ring
23
Q
Fosfomycin inhibits
A
Enzyme at first step of cell wall synthesis
24
Q
Gylcopeptides/lipoglycopeptides types
A
- Vancomycin
- Teicoplanin
25
Monobactam types
Aztreonam
26
Desirable properties of agents
* Non-toxic; limited side-effects
* Able to enter cell
* Specific target
* Sufficient concentration
* Spectrum
* Soluble in body fluids; remain active
* Limited development of resistance
27
Beta lactams share a common
Beta-lactam rings
28
Bata-lactams principle mode of action
Inhibition of cell wall synthesis
29
Additions to the beta-lactam ring determine
whether the agent is a penicillin, cephem,
carbapenem, or monobactam.
30
Commonly used beta-lactum ring agents are due to their
broad spectra
of antibacterial activity, safety profiles, and proven
clinical efficacy
31
if the drug name begins with ceph or cillin, or penem
it is a beta lactam
32
Beta-lactam ring is similar to
* Structurally similar to acyl-D-alanyl-D-
alanine (substrate for linear
glycopeptide in cell wall)
33
Beta lactam ring binds to
Bind to penicillin binding proteins
(PBPs)causing cell death
* PBPs cross-link cell walls
* PBPs essential for survival
34
all bacteria have pores and they are called
Porins or small pores
35
Glycopeptides/ Lipoglycopeptides are for what type of bacteria
gram positive bacteria
36
Glycopeptides- Vancomycin and teicoplanin what molecules and do not
* Large molecules
* Do not bind PBPs
37
Glycopeptides ( Vancomycin and Teicoplanin) bind
to precursors of cell wall synthesis
* Form a hydrogen bond with terminal D-alanyl-D-
alanine moiety of NAM/NAG peptides.
* Interferes with ability of PBP to incorporate precursors into cell wall
38
Lipoglycopeptides( oritavancin, Dalbavancin, Telavancin are Structurally similar to
vancomycin—
have addition of hydrophobic group
39
Lipoglycopeptides( oritavancin, Dalbavancin, Telavancin)
-Structurally similar to vancomycin—
have addition of hydrophobic group
Allows for binding to cell membrane—
increasing inhibition of cell wall
synthesis
* Also, increase permeability and
polarize cell membrane potential
* Can be affective for Vancomycin
Intermediate Staphylococcus aureus
(VISA).
40
Vancomycin mechanism of Action
Inhibits cell wall synthesis by binding to the D-ala-D-ala terminal of the
growing peptide during cell wall synthesis
41
Vancomycin use
* For treatment of serious infections caused
by β-lactam-resistant gram-positive
microorganisms or if patient is allergic to β-
lactam drugs
42
Vancomycin use for Prophylaxis for
endocarditis or
implantation of prostheses. Prophylaxis
should be discontinued after a maximum of
two doses.
43
Vancomycin use for what if unresponsive to what
for Pseudomembranous colitis if unresponsive to metronidazole
44
Vancomycin use is discouraged if
* Routine surgical prophylaxis
* Empiric therapy (unless evidence of Gram
-positive infection or prevalence of MRSA
is high)
* Treatment of a single positive blood
culture
* Continued use after susceptibility report
* Eradication of MRSA colonization
45
Inhibitors of cell membrane function
Polymyxin and Lipopeptides
46
Polymyxin description
* Cyclic lipopeptides
* Polymixin B and Colistin
* Act as detergents (interacting with phospholipids)
* Results in leakage of macromolecules from bacterial cells
* Also human cell membranes
* Toxicity issues (neurotoxicity and nephrotoxicity)
47
Polymyxin acts like what
A detergent( Interacts with phospholipids)
- results in leakage of macromolecules from bacteria cells
48
Polymyxin are what lipopeptides
Cyclic
49
What are the types of Cyclic lipopeptides
Polymixin B and Colistin
50
How is Polymyxin toxic
Damage cell membranes
- Toxicity includes Neurotoxicity and Nephrotoxicity
51
Daptomycin can Bind to and
* Binds to and disrupts Gram positive cell membrane
* Inserts hydrophobic tail into membrane
* Increases permeability, killing the cell
52
Daptomycin is what type
Lipopeptides
53
Polymyxin B can combined with
neomycin and bacitracin
as a topical antimicrobial
preparation
54
Colisitin was used between
1952 and 1980 for Gram negative rod infections
55
Colistin causes in humans
Renal toxicity and replaced by other antibiotics with less toxicity
56
Colistin is used for
Multi-drug resistant GNR and is a last line of defense
57
Daptomycin (DAP) is potent against
Gram postive cocci including resistant strains such as MRSA (Methicillin Resistant Staphylococcus
aureus) and
* VRE (Vancomycin Resistant Enterococcus sp.
58
Daptomycin large size prevents
it from penetrating gram negative organisms outer membrane
59
Daptomycin is not useful in
treatment of respiratory infections
- lung surfactant binds the drug, inactivating it
60
Inhibitors of protein synthesis
* Aminoglycosides
* Macrolide-lincosamide-
streptogramin (MLS)
* Ketolides
* Oxazolidinones
* Chloramphenicol
* Tetracyclines
* Glycylglycines
61
Amino-glycosides types
tobramycin, gentamicin, amikacin
62
Aminoglycosides are
broad spectrum
63
Aminoglycosides inhibit protein synthesis by
* Irreversibly binds protein receptors on bacterial 30S
ribosome
64
Amino-glycosides are used with and have an increased
* Often used with beta-lactam (synergistic effect)
* Bacterial uptake increased
65
Aminoglycosides inhibit what bacteria
* Wide variety of GN organisms and certain GP –e.g. Staph.
aureus
66
Aminoglycosides can not inhibit what
* Anaerobic bacteria cannot take these agents up intracellularly –
so typically not active
67
What should be monitored during therapy with Amino-glycosides
* Blood levels must be monitored during therapy
- Nephrotoxicity
- Auditory and Vestibular Toxicity
68
Macrolide group
Macrolide=
Macrolide-lincosamide-streptogramin
* Macrolide=erythromycin, azithromycin,
clarithromycin
69
The macrolide group is generally what activity
* Generally bacteriostatic, but can be
bactericidal if infective dose is low and drug
concentration is high
70
The Macrolide group binds to
* Binds the 23S ribosomal RNA on the 50S
ribosomal subunit
71
The Macrolide group disrupts what and has uptake difficulties with what
* Disrupts the growing peptide chain by
preventing translocation
* Uptake difficulties with G- bacteria
72
Lincosamide type
Clindamycin
73
Clindamycin binds the blank and interferes with blank
50s subunit ribosomal subunit
- prevents elongation ( Interferes with peptidyl transfer)
74
Clindamycin has what activity
gram positive activity and is Bactericidal or bacteriostatic
-* Activity against anaerobic Gram positive and
some anaerobic Gram negative
75
Clindamycin is associated with an increased risk of
C.diff infections
76
Streptogramin-
Quinupristin/dalfopristin are what
(Synercid)
1. Synergistic (either is static; combination is cidal
77
Streptogramin-
Quinupristin/dalfopristin 1st step
First, dalfopristin binds to the ribosomal
50S unit changing the conformation of the
ribosome.
78
Streptogramin-
Quinupristin/dalfopristin 2nd step
-Increasing the affinity of quinupristin that
in turn binds to the bacterial ribosome
-This double binding interrupts protein
synthesis and blocks bacterial growth
79
Oxazolidinones types
* Linezolid (trade name = Zyvox) and tedizolid
* Relatively new class of antibacterial agents
- Synthetic
80
Oxazolidinones inhibit protien synthesis through
protein synthesis through a unique
mechanism
* Binds the 23S ribosomal RNA of the 50S
subunit, prevents the formation of a
functional 70S initiation complex. This
prevents bacterial translation and replication.
* New mechanism=no cross resistance with other drugs
81
Oxazolidinones inhibit what organisms
* Gram positives and Mycobacteria
82
Chloramphenicol action, inhibits what organisms, and are is associated with what
* inhibits protein synthesis by binding the 50S subunit—inhibits
transpeptidation
* Gram negative and Gram positive activity
* Toxicity (Bone marrow—aplastic anemia)
83
Tetracycline/ Doxycycline is a
broad spectrum antibiotic
- bacteriostatic
- toxic to upper GI and causes cutaneous phototoxicity and Photoallergenic immune reactions
84
Tetracycline/ Doxycycline binds to and inhibits what organism
* Bind 30S subunit; incoming tRNAs –
amino acid complexes cannot bind to
the ribosome.
* G+, G-, intracellular bacterial
pathogens
85
Glycylglycines are
* Semisynthetic tetracycline derivative
* Tigecycline
- have GI side effects
86
Glycylglycines inhibit what and are not affected by what
Inhibits protein synthesis
* Reversibly binds the 30S ribosomal subunit
* Not affected by most common
tetracycline resistance mechanisms
87
Inhibitors of DNA and RNA synthesis
* Fluoroquinolones
* Metronidazole
* Rifampin
88
Fluoroquinolones bind to and interfere with
Bind to and interfere with DNA gyrase and topoisomerase
(involved in bacterial supercoiling)
89
Fluoroquinolones inhibit what organisms, human side effects, and are what mode of action
* Bactericidal
* Broad spectrum-Gram positive and Gram negative,
but resistance has developed.
* Side effects = affects in tendons (Tendonitis and rupture)
90
types of Fluoroquinolones
* Nalidixic acid= older drug
Ciprofloxacin, levofloxacin,
Ofloxacin, norfloxacin (UTI),
Moxifloxacin
91
Metronidazole disrupts what, requires what atmosphere, and is potent against
* Disrupts helical structure of DNA
* Activation requires reduced
atmosphere (anaerobic)
* Most potent against anaerobes
and microaerophilic organisms
92
****Metronidazole kills
* kills both C. difficile
* Also Trichomonas and other
amoebae
93
Rifampin characteristics
* Semisynthetic
* Binds RNA polymerase; inhibits
synthesis of RNA
* Better for Gram positive
94
Rifampin develop what, has what type of mutations, and is used with
* Develops resistance quickly
* Spontaneous mutations---rifampin-
insensitive RNAP
* Used in combination with other
drugs
95
Inhibitors of Other Metabolic
Processes
* Sulfonamides
* Trimethoprim
* Nitrofurantoin
96
Sulfonamides characteristics
* Also disrupts bacterial folic acid pathway
(different enzyme inhibited)
* Active against wide variety of Gram
positive and negative except P.
aeruginosa
97
Trimethoprim
* Disrupts folic acid pathway
* Can combine with sulfonamide for better
activity
* Active against several Gram positive and
negative except P. aeruginosa
98
Trimethoprim binds to dihydrofolate reductase to inhibit
Dihydrofolic acid ----> Tetrahydrofolic acid ( active form of folic acid)
99
Sulfonamide inhibits what enzyme
Para-aminobenzoic acid
100
Nitrofurantoin characteristics
* Activated by bacterial flavoproteins
(nitroreductase). Reduced reactive intermediates
damage ribosomes and other macromolecules such
as DNA, RNA and proteins.
* Good activity for most Gram positive and Gram
negatives that cause UTI (not P. aeruginosa)
* Only for urinary tract infections.
101
Clinical Resistance
loss of antimicrobial
susceptibility to the extent that the agent is no
longer effective for clinical use.
102
Microorganism- mediated antimicrobial resistance
– due to genetically encoded traits
Where a bacteria transfers resistance to another bacteria
103
Examples of intrinsic resistance
1. Pseudomonas: poor diffusion through
cellular envelope and efflux pumps
2. Vancomycin cannot penetrate gram-
negative cell wall
3. Enterococcus: cephalosporins cannot bind
PBPs
4. All Klebsiella pneumoniae produce a
beta-lactamase that inactivates ampicillin
104
Anaerobic bacteria versus aminoglycosides
* Lack of oxidative metabolism to drive uptake of
aminoglycosides
105
Gram-positive bacteria versus aztreonam (beta-lactam)
* Lack of penicillin-binding protein (PBP) targets that bind
106
Gram-negative bacteria versus vancomycin
* Lack of uptake resulting inability to penetrate outer
membrane
107
Pseudomonas aeruginosa versus sulfonamides,
trimethoprim, tetracycline, or chloramphenicol
* Lack of uptake - ineffective intracellular concentrations
108
Klebsiella spp. versus ampicillin (a beta-lactam) targets
* Production of enzymes (beta-lactamases) that destroy
ampicillin before it reaches its PBP target
109
Aerobic bacteria versus metronidazole
* inability to anaerobically reduce drug to its active form
110
Enterococci versus aminoglycosides
* Lack of sufficient oxidative metabolism to drive uptake of
aminoglycosides
111
Enterococci versus aminoglycosides
* Lack of sufficient oxidative metabolism to drive uptake of
aminoglycosides
112
Enterococci versus all cephalosporin antibiotics
* Lack of PBPs that effectively bind
112
Enterococci versus all cephalosporin antibiotics
* Lack of PBPs that effectively bind
113
Lactobacilli and Leuconostoc spp. versus vancomycin
* Lack of appropriate cell wall precursor target
114
Lactobacilli and Leuconostoc spp. versus vancomycin
* Lack of appropriate cell wall precursor target
115
Stenotrophomonas maltophilia versus carbapenems (beta
-lactam)
* Production of enzymes (beta-lactamases) that destroy
carbapenem before it reaches PBP targets
116
Acquired Resistance characteristics
* Mutations
* Horizontal gene transfer:
* Transformation
* Transduction
* Conjugation
* Often Plasmid mediated
Important:
* We test for acquired
resistance not intrinsic
resistance.
117
Frequent Methods of Resistance
* Enzymatic degradation
* Decreased uptake
* Increased efflux
* Altered target
117
Frequent Methods of Resistance
* Enzymatic degradation
* Decreased uptake
* Increased efflux
* Altered target
118
Resistance to Beta Lactams (penicillins,
cephalosporins, carbapenems, etc.)
1.) Enzymatic destruction ( Beta- lactamases)
* Most common method of resistance
* Thousands of different types of beta-lactamases
* Some beta-lactamases are encoded on mobile genetic
elements (e.g. plasmids) and others are encoded on
chromosomes.
119
Resistance to Beta Lactams (penicillins,
cephalosporins, carbapenems
Class A subset a - Extended spectrum beta lactamases
* Significant impact clinically
* Confers resistance to penicillin, cephalosporins, and
aztreonam
* Derived from the narrow-spectrum beta-lactamases
(TEM-1, TEM2 or SHV-1, OXA, CTX-M and GES
ensyme)
* Klebsiella, E. coli and enterobacteria
120
Resistance to Beta Lactams (penicillins,
cephalosporins, carbapenems
class A subset b. Carbapenemases
* High level clinical impact
* Aztreonam is variable depending on the genotype
CRE, CRO, MDRO
* Serine beta-lactamase
* KPC (Klebsiella pneumonia carbapenemases: Resistant to all
penicillins, cephalosporins, carbapenems, and aztreonam
121
Resistance to Beta Lactams (penicillins,
cephalosporins, carbapenems
Class B
-Metallo-beta-lactamases (MBLs) requires zinc at active
site
-to hydrolyze all beta-lactams NDM, IMP and VIM
122
Resistance to Beta Lactams (penicillins,
cephalosporins, carbapenems
Class C
AmpC: hydrolyze most cephalosporins except cefipime,
cephamycin (cefoxitin, cepotetan), aztreonam and penicillin.
123
Resistance to Beta Lactams (penicillins,
cephalosporins, carbapenems
Class D
Oxacillinases (OXAs) OXA 48 and OXA 23 like. Resistant to
all penicillins, cephalosporins, carbapenems, and
aztreonam
124
2. Altered antibiotic target (mutations in PBPs)
Lower affinity binding
mecA confers resistance in Staphylococcus sp. and
Streptococcus sp. (e.g. MRSA)
125
MRSA
* Methicillin resistant Staphylococcus aureus
125
MRSA
* Methicillin resistant Staphylococcus aureus
126
MRSA what is tested, other characteristics
* Cefoxitin resistance can be used as a screen
* PBP 2A latex agglutination tests
* PCR to detect genes
* mecA – most common
* mecC – newer variant identified in England
* Limited data on prevalence in the US
127
Beta-Lactam/Beta Lactamase Inhibitor
Drugs mode of actions
* Inhibitor binds beta-lactamase allowing, beta-lactam to
work
* Older generation were suicide inhibitors
* Most have no intrinsic antibacterial activity
* Exception - Sulbactam is active against Acinetobacter
128
Beta-Lactam/Beta Lactamase Inhibitor
Drugs examples
* Amoxicillin/clavulanic acid
* Ampicillin/sulbactam
* Piperacillin/tazobactam
129
Vancomycin Resistance
* VRE – Enterococcus
* vanA, vanB, vanC, vanD, and vanE
129
Beta-Lactam/Beta Lactamase Inhibitor
Drugs newer generation
* Ceftolozane/tazobactam
* Ceftazidime/avibactam
130
Vancomycin Resistance
Intrinsic
* Enterococcus casseliflavus/flavescens and
gallinarum
* Low-level
131
Vancomycin Resistance
* Acquired resistance (vanA most common)
* Enterococcus faecalis, faecium, raffinosus,
avium, and durans
* Altered target - Alteration in the molecular
structure of cell wall precursor components
decreases binding of vancomycin, allowing cell
wall synthesis to continue.
132
Aminoglycoside Resistance
Enzymatic
* Modification of aminoglycoside
* Decreased affinity to bind the 30S ribosome
* Allows translation to continue
133
Aminoglycoside Resistance
Altered target
* Mutations in ribosomal targets (rare)
134
Aminoglycoside Resistance
Decreased uptake
* Porin (OMP) mutations
135
Fluoroquniolone resistance
* Decreased uptake
* Altered target
* Mutations in DNA gyrase or
toposiomerase
* Efflux
136
Resistance to Trimethoprim
* Bacteria need to make their own folic acid
* Use a vital pathway that involves the
enzyme dihydrofolate reductase.
* Trimethoprim inhibits this bacterial
enzyme
* some bacteria bypass this step by acquiring a
new enzyme that bypasses the old, inhibited
dihydrofolate reductase.
* The new enzyme comes from (you guessed it)
plasmids.
137
Goals of tests
* Determine the best option for treatment
* Relevant testing
Standardization
* Environmental factors must be minimized
* Optimize growth conditions
* Optimize antimicrobial integrity
* Maintain reproducibility/consistency
138
In vitro standardization
Bacterial Inoculum
0.5 Macfarland
139
In vitro standardization
medium ( Mueller Hinton agar or broth
* agar depth ~4mm
* Cation concentration – too high can cause a decrease in
zone sizes
* pH – neutral ~7.4
* Thymidine concentration – should not interfere with
detection of tetracycline and sulfonamide resistance
(can cause inhibition if concentration to high)
140
In vitro standardization
Incubation time and temperature
35 C, 16-18 hours
141
In vitro standardization
Concentrations of antimicrobial agent
* Follow CLSI concentrations or FDA package insert
concentrations
142
Inoculum Standardization
A 0.5 McFarland Standard contains
about 1.5 x 108 CFU/mL (used for disk diffusion and E-test)
Broth dilution methods are standardized using
5 x 105 CFU/mL.
143
media
Mueller Hinton broth
* Purchased commercially
* pH, cation concentration, thymidine content controlled by
manufacturer
144
media
NaCL is needed for
MRSA
145
media
Blood MH is needed for
Viridans Streptococcus or Streptococcus
pneumoniae
146
media
Chocolate MH is needed for
N. meningitidis
147
media
Haemophilus test medium is used for
Haemophilus
148
Incubation requirements
* After test is set up, DO PURITY CHECK
* 350, non-CO2
* Can use CO2 for fastidious (Neisseria, Haemophilus,
streptococci)
149
Microbroth dilution
* Usually MH broth
* Serially dilute antimicrobial agent in tubes or
wells
* Add organism suspension: final
concentration is 5 x 105 (dilution of 0.5
McFarland)
* Incubate
* Read MIC
150
Agar dilution
* Incorporate antimicrobial agent in semi-liquid agar
at varying concentrations
* Inoculate bacterial suspensions on surface of agar
* Incubate
* Read MIC
151
Disk diffusion ( Kirby Bauer)
* 0.5 McFarland
* MH Media
* Can test multiple drugs
* Use antimicrobial agent impregnated disks
* Agent diffuses into agar: concentration gradient
* Incubate
* Measure zone diameters, correlate to S, I, R
* No MICs
* Storage of disks
* Must be stored at proper temperature
* Frequently requires -20C
152
Antimicrobial Susceptibility Test
Methods: Interpretive Breakpoints
- Establishing zone
diameter interpretive
breakpoints
- Hundreds of samples
tested and zone of diameter
correlated with MICs to get
ranges
153
the E test
* 0.5 McFarland
* MH Media
* Gradient diffusion
* Antibiotic concentration changes along the strip
* Can obtain MIC
154
Advantages and disadvantages of broth dilution
* A: can obtain MIC
* D: amt. of tubes/pipetting, time consuming, not as
reproducible
155
Advantages and disadvantages of micro dilution
* Microdilution
* A: better reproducibility, ease of inoculation, can test
many drugs at once
* D: skipped wells, expensive, less choice of drugs
156
Advantages and disadvantages of Kirby Bauer
* A: easy and cheap, more choices of drugs
* D: lots of variables
157
Advantages and disadvantages of E test
* A: ease of use and reading, can test organisms
that don’t grow well in broth systems
* D: more expensive
158
types of automated AST
-Vitek
- microscan
159
Vitek
* Small card
* Microdilution in tiny wells
* Algorithm based MICs generated by software
* Cannot see what is happening
* 8-24 hour results
* Has more limitations because of algorithm
160
Microscan
* Microdilution in microtiter plate
* Lyophilized version of reference method
* Can be read by instrument or manually
* 16-48 hour results
161
QC
* Daily/weekly
* IQCP (Individualized Quality Control Plan)
required for weekly testing
* New lot/new test
* Use ATCC strains
* QC strains are found in package insert or CLSI
Manual (M100)
162
Common test battery
Enterobacteriaceae
* Beta lactams including: ampicillin, cephalosporins, and
carbapenems; aminoglycosides; fluoroquinolones
163
Common test battery
Pseudomonas
* Other beta lactams such as Piperacillin, ceftazidime(3rd
generation cephalosporin) and cefepime (4th generation
cephalosporin), and carbapenems), and aminoglycosides,
fluoroquinolones
164
Common test battery
Staphylococcus
* Pen & Amp, erythromycin, clindamycin, fluoroquinolones,
vancomycin, doxycycline, trimethoprim/sulfa.
165
Clinical reporting
* CLSI has recommendations for reporting by
drug/bug combo
* FDA indications also important
* Body site and infection specificity
* Not all drugs are cleared to treat all types of
infections in all sites
* Reporting should be determined by
laboratory with input from Pharmacy and
Infectious Disease Physicians
166
Antibiogram
* Provides guidance for empiric treatment
* Provides guidance on resistance patterns in a particular
hospital or system
167
Alternative Approaches for MRSA
Oxacilin agar screen
* Technique to ensure detection of heteroresistant
populations (MSSA and MRSA)
* In conventional testing: 5% NaCl, 35o, 24 hours
168
Alternative Approaches for MRSA
PBP2a detection
* Detect using latex agglutination or
immunochromatographic membrane tests
169
Alternative Approaches for MRSA what molecular test
* PCR
* Detect mecA gene
170
Oxacillin screen agar
* -Resistance by penicillin binding proteins (PBP) via mecA gene
* -Test oxacillin in lab (more stable) - if resistant, all
cephalosporins are reported as resistant
* -Newer method uses cefoxitin for greater sensitivity (usually
cefoxitin disk)
171
Oxacillin screen agar
* -Resistance by penicillin binding proteins (PBP) via mecA gene
* -Test oxacillin in lab (more stable) - if resistant, all
cephalosporins are reported as resistant
* -Newer method uses cefoxitin for greater sensitivity (usually
cefoxitin disk)
172
Oxacillin disk screen
– screen for
Streptococcus pneumoniae ( penicillin resistance)
173
Enterococci screening test
* Aminoglycoside screen – tests synergy of
two drug classes
* Synergy: Ampicillin + Gentamicin
* Test for VRE (vancomycin agar screen)
174
ChromAgar
Screening agar – used for determining MRSA
carriage in the nares
175
Beta-lactamase
* Use chromogenic cephalosporin (nitrocefin,
cefinase)
* Use for Staphylococcus, N. gonorrhoeae, H.
influenzae, enterococci, sometimes
Bacteroides sp.---NOT for enteric GNRs
176
ESBL
* Extended Spectrum Beta Lactamase
* E. coli, K. pneumoniae and others
* In vitro susceptibility not reliable for B-lactam
drugs
* Test using B-lactam inhibitor
***New Cephalosporin Breakpoint Released by CLSI in
2010 made ESBL testing no longer required for Clinical
Reporting***
177
ESBL Testing (rarely used now)
Look for ≥ 5 mm zone increase
CAZ, CAZ/CLA
CTX, CTX/CLA
178
ESBL confirmation
-Can also use broth dilution:
-Look for ≥ 3 doubling dilution decrease with either
drug:
-e.g. ceftazidime MIC = 8 ug/mL
ceftazidime/clavulanate MIC = 1 ug/mL
-This is the basis for confirmation FDA-approved
commercial tests
179
Reporting ESBL test
* Report resistant for all penicillins, cephalosporins,
and aztreonam regardless of in vitro status
* Not cephamycins (cefoxitin/cefotetan)
180
Detection methods for
Carbapenemases
* Modified Carbapenem Inactivation Method (mCIM)
* Incubate organism in water with 10ug disk of
meropenem for 2 hours
* Place disk on lawn of susceptible E. coli and look for
presence or absence or zone of clearing
181
Detection methods for
Carbapenemases
CarbaNP
* Hydrolysis methods which detect carbapenem
degradation products – color change
182
Detection methods for
Carbapenemases
* Lateral flow immunoassays
* Detect carbapenemase enzymes using specific
antibodies
183
Detection methods for
Carbapenemases. Molecular methods
PCR) – detects genes
184
Detection methods for
Carbapenemases
Modified Hodge test is no
no longer recommended
185
D test
* The erm gene product confers clindamycin
resistance in S. aureus.
* Clindamycin resistance is INDUCIBLE
* Any Staphylococcus Or Beta Streptococcus that
tests susceptible to clindamycin and NOT
susceptible to erythromycin must have a D-Test
performed
186
Clindamycin Induction
* To determine if S.
aureus/MRSA is
susceptible to Clindamycin
* S. aureus/MRSA resistant
to erythromycin and
susceptible to clindamycin
have to be tested
* Zone around clindamycin
disk will be blunted to
form D if clindamycin can
be induced by
erythrothromycin to be
resistant
187
Limitations of Susceptibility/Resistance
Testing
* In vitro/in vivo
* Phenotypic vs. Genotypic
* Diffusion of agent in tissues
* Patient status
* Virulence and pathogenicity of
organism
* Site & severity of infection
