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Learning Outcomes
At the end of the session the student should be able to:
Antimicrobial Resistance 1. Define antimicrobial resistance
2. List potential contributors to antimicrobial resistance
Professor Patrick Akpaka 3. Describe the different Mechanisms of antimicrobial
Dept. of Paraclinical Sciences resistance
4. Discuss measures to control antimicrobial resistance
The University of the West Indies
5. Describe methods of detecting antimicrobial resistance
St. Augustine 6. Explain how to monitor antimicrobial use in healthcare
facilities – surveillance and stewardship
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Sir Alexander Fleming & Discovery of Antibiotics

Compounds which Introduction
Soluble substances
destroy microbes,
derived from a
prevent their Versus
mould or bacterium
multiplication or ANTIMICROBIAL ANTIBIOTIC that inhibits the
growth, or prevent
growth of other
their pathogenic
microorganisms Fleming’s Petri Dish
especially bacteria
•Initial plate of Staphylococci plate contaminated with Penicillium notatum
• What was there before antibiotics? (now known as Penicillium chrysogenum)
• How were infectious diseases cured? Treated? •Around the fungal colony is a clear zone where no bacteria are growing
•Zone of inhibition due to the diffusion of a substance with antibiotic
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9/6/2017 from the fungus 4

Fleming & The Discovery of Antibiotics… Penicillin discovery

• This first practical
use of penicillin was  Penicillin was isolated in
in the preparation 1939, and in 1944
of differential Selman Waksman and
culture medium. Albert Schatz, American
microbiologists, isolated
• The amazing thing
Streptomycin and a
that caught our
number of other
attention is, Fleming
antibiotics from
had already
Streptomyces griseus.
highlighted this
issue of AMR
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Antibiotics discoveries timeline

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Features of Antimicrobial Drugs

 Most modern antibiotics come from species of
microorganisms that live in the soil
 To commercially produce antibiotic:
1. Select strain and grow in broth
2. When maximum antibiotic concentration
• From the time a baby is born, he/she is exposed to myriads reached, extract from medium
of bacteria especially Staphylococcus aureus 3. Purify
• Some will be pathogenic or just exists as normal flora 4. Chemical alter to make it more stable
• We have to be mindful of these when lab results

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Growth phase of microorganisms Selective Toxicity and Route of administration

• Cause greater harm to microorganisms than to host
When is the best time to • Chemotherapeutic index = lowest dose toxic to patient
Stationary phase
administer antimicrobial divided by dose typically used for therapy
Log of # of Logarithmic phase
bacteria/mil Death phase

Lag phase

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Features of Antimicrobial Drugs:

Spectrum of Activity
Antimicrobial Action
 Bacteriostatic - inhibit or stop growth of microorganisms
• Antimicrobial medications vary with respect to
 Bactericidal – kill cells No antibiotic the range of microorganisms they kill or inhibit
Bacteristatic • Some kill only limited range : Narrow-spectrum
• While others kill wide range of microorganisms:
Number of bacteria

Bactericidal Broad-spectrum antimicrobial

Abx added

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Spectrum of Action
Effects of Combining Drugs
• The number of different kinds of pathogens a
drug acts against.
• Combinations are sometimes used to fight infections
• Narrow Spectrum and Broad-Spectrum Drugs
• Synergistic: action of one drug enhances the activity of
• Antagonistic: activity of one drug interferes with the
action of another
• Additive: neither synergistic or antagonistic
 Indifference: Each drug works no better or no worse
alone or in combination

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Adverse Effects Selection of Antibiotic

• Requires clinical judgment, detailed knowledge of
1. Allergic Reactions: some people develop hypersensitivities
pharmacological and microbiological factors.
to antimicrobials
• Empirical therapy – initial – infecting organism not
2. Toxic Effects: some antimicrobials toxic at high
identified – single broad spectrum agent
concentrations or cause adverse effects
• Definitive therapy- microorganism identified – a
3. Suppression of normal flora: when normal flora killed,
narrow –spectrum low toxicity regiment to complete
other pathogens may be able to grow to high numbers
the course of treatment

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PK/PD for effective agents and dosage

Antibiotic Selection contd.
• Knowledge of the most likely infecting
microorganism and its susceptibility
• Gram stain; Pending isolation and identification of
the pathogen
• Specimen for culture from site of infection should be
obtain before initiation of therapy PK/PD determinants
• Definite therapy  T for which non protein bound conc. exceed MIC (T>MIC)
 Ratio of Peak conc. [Cmax] and MIC; [Cmax / MIC]
 Drug exposure and MIC
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Antiretroviral therapy (ART) is typically used

Mechanisms of action of Antibacterial Drugs for the treatment of HIV.

The targets of drug classes

currently in use are shown here.
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Antifungal drugs target several different cell

1. Inhibitors of Cell Wall Synthesis
(a)β-lactam antibiotics : Inhibit the transpeptidation
enzymes essential in the manufacture of
(b) Cycloserine: Is an analog of D- analine and also
interferes with peptidoglycan synthesis. It blocks the
action of alanine racemase, an important enzyme in
the incorporation of D- alanine in the pentapeptide of
(c) Vancomycin, Teicoplanin, Bacitracin, Novobiocin:
Inhibit the early stages in peptidoglycan synthesis.
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Beta-Lactam Structure
Resistance to Antimicrobials

• Some microorganisms inherently resistant

to effects of a particular drug
• Other previously sensitive microorganisms
can develop resistance through
spontaneous mutations or acquisition of
new genes (genetic transfer)
Imp, Mem, Erta, Dori.

Some bacteria produce b-lactamase- enzyme that breaks the critical b-lactam
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Mechanisms of Action of Antibiotics Definition of Antimicrobial Resistance

• The WHO describes Antibiotic Resistance as ‘’one of

Mechanisms Antimicrobial target Antimicrobial family
the greatest threats to human health ‘’
of action of Cell Wall Beta-lactams and vancomicin
the most Daptomicin, Polimixin • Antimicrobial Resistance is defined as a situation
Cellular membrane
frequently where microorganisms continue to multiply although
Inhibiting Protein synthesis Linezolid, Tetraciclins, Macrolides,
used family of Aminoglicosides
exposed to antibiotic or antimicrobial agents.
antibiotics. Inhibiting Synthesis of DNA/RNA
Fluoroquinolones, Rifamicin • Antimicrobial drug resistant bacteria Vs Multidrug
Targeting Metabolic Pathways resistant bacteria? (MDR, XMDR)
Trimethoprim, Sulfonamides

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Definitions: MDR, PDR & XDR Facts about AMR (CDC)

• On consumption of antibiotics, sensitive bacteria are killed, but
• MDR: Multi-drug resistant strains” are resistant to at least 3 resistant ones may be left to grow and multiply.
classes of the following 5 drug classes:
• Overuse and misuse of antibiotics threatens the usefulness of these
1. Antipseudomonal cephalosporins
important drugs. Decreasing inappropriate antibiotic use is a key
2. Antipseudomonal carbapenems
strategy to control antibiotic resistance.
3. β-lactam–β-lactamase inhibitor
NEJM 2008;358:1271-81; • Antibiotic resistance can cause significant suffering for people who have
4. Fluoroquinolones Clin Micro Rev 2008, 21(3):538–582 common infections that once were easily treatable with antibiotics.
5. Aminoglycosides Emerg Infect Dis 2009, 15(6): 980-981
• When antibiotics do not work, infections often last longer, cause more
• PDR: “Pan-drug resistant strains” are resistant to all standard
antimicrobial agents tested except colistin severe illness, require more doctor visits or extended hospital stays, and
involve more expensive and toxic medications.
• XDR: “Extensively drug resistant strains” are resistant to all
commercially available antimicrobial drugs, including colistin • Some resistant infections can even cause death.
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SEPTEMBER • CDC report: Antibiotic Resistance

2013 Threats in the United States

Figure :Timeline of • Assessment of domestic antibiotic resistance threats

antibiotics discovery and • C. difficile
the development of
antibiotic resistance • Carbapenem-resistant Enterobacteriaceae (CRE)
• Drug-resistant Neisseria gonorrhoeae

MDR Acinetobacter; DR Campylobacter

ESBLs; VRE; MRSA; MDR P. aeruginosa
Strep pneumoniae; Fluconazole-resistant Candida;

• Erythromycin-resistant Streptococcus Group A
Antibiotics and their journey from discovery to resistance • Clindamycin-resistant Streptococcus Group B
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CDC Antibiotic Resistance Threats in the United States, 2013 .

1. Overuse
 As early as 1945 Sir A Fleming raised alarm regarding antibiotic
overuse (New York Times 6/26/45). Misuse of penicillin could lead
to propagation of mutant forms of bacteria that would resist the

Causes of Antimicrobial new miracle drug. Ca.14 % of Staph strains isolated in a London
hospital had developed resistance to penicillin by 1946
 Antibiotic consumption = Emergence of resistant strains
agents resistance crisis  Selective pressure
 Over the counter antibiotic prescriptions
 Patient demands
 Not completing prescribed courses
Nature, 2013; 495(7440): 141
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2. Inappropriate prescribing 3. Extensive agricultural usage

• Antibiotics used as growth supplements in livestock
• Studies have shown that indication, choice or duration of • Estimated 80% of antibiotics used in animals in the US
antibiotic treatment is incorrect in 30-50% of cases (Luyt • Improve animal health, produce larger yields, and
et al, Crit Care 2014, 14(8): 480 higher quality products
• Sub-inhibitory and sub-therapeutic antibiotic • Abx used in livestock ingested by humans when
concentrations can promote the development of consuming food
antibiotic resistance

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4. Environmental usage Modes of transmission of resistance

• Agricultural use affect the environment as Conventional modes
antibiotics are excreted in urine and stool by • Health Care Workers (HCW) hands and
animals objects
• Tetracyclines and streptomycin used for Molecular modes
spraying fruit trees • Conjugation, Transduction, Transformation
• In sprays used against insecticides, house holds
utensils etc.

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There’s always a new kid in MECHANISMS OF RESISTANCE

block – KPC, NDM, CRK 1. Enzymatic inhibition
2. Alteration of bacterial membranes
Outer membrane permeability
Inner membrane permeability
3. Rapid ejection of the drug [efflux] or reduced drug influx.
4. By pass of antibiotic inhibition.
5. Alteration of target sites
Altered ribosomal target sites
Altered cell wall precursor targets
Altered target enzymes
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Molecular genetics of antibiotic resistance
Genetic variability is essential for microbial evolution. It may occur in
a variety of ways :-
oMicro evolutionary changes by point mutations-in nucleotide
base pair
oMacro evolutionary changes [whole scale changes] like
-Inversions, Duplications, Deletions, Transposition
oAcquisition of foreign DNA –plasmid mediated, bacteriophage,

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b-Lactam Drugs
1.Enzymatic inhibition
 Some bacteria
Enzymes inactivating antibiotics
produce b-
 Beta-lactamases-split amide bond of the lactamase-
beta lactam ring.
enzyme that
 There are many types-characterised by
amino acid and nucleotide sequencing breaks the
 Class A MWT 29000-Preferentially hydrolyze critical b-lactam
penicillins e.g. TEM-1 prevalent in many gram neg
 Class B-metalloenzymes have a zinc –binding thiol
 b-lactam drugs
group required for beta lactamase activity
 Class C mwt 39000 –mainly cephalosporinases
 Class D-oxacillin –hydrolyzing enzymes include: pen,
Many beta lactamases are plasmid mediated all are
produced constitutively there are 6 main groups
1. Those that hydrolyze benzylpenicillin carbapenems
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Beta lactamases Modifying enzymes

1. Those that hydrolyze oxacillin and related penicillins  Reactions are-
2. Carbenicillinases N-acetylation
3. Those that break extended spectrum beta lactams like aztreonam
4. Those that break down oxyimino B lactams
O nuleotidylation
5. Carbapenemases-found in pseudomonas O phosphorylation
Most cephalosporinases are inhibited by clavulanate,sulbactam or  Chloramphenicol acetyltransferase-inactivates the drug by 3-
tazobactam. Carbapenamases are metalloenzymes inhibited by EDTA o-acetylation-plasmid mediated/chromosomal
but not clavulanate or sulbactam
 Erythromycin esterase-seen in E coli-hydrolyze lactone ring
Production of enzymes modifying antibiotics
thus deactivating it-limits utility of oral erythromycin in
 . Aminoglycosides, chloramphenicol-coded by plasmids or chromosomal reducing the aerobic gram neg flora of the GIT prior to Gi
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2. Alteration of bacterial membranes Alteration of bacterial membranes contd.

 Outer membrane permeability—outer membrane of
gram neg acts as a barrier to antibiotics esp hydrophobic • Promotion of antibiotic efflux-major mechanism for
ones. tetracycline resistance in gram negative
 Inner membrane permeability- rate of entry of plasmid/chromosomal/transposoon mediated
aminoglycosides into bacterial cells is a function of them
binding to a non saturable anionic transporter,where they
retain their positive charge and are pulled across the
cytoplasmic membrane by the internal charge of the
cell.This is an energy dependent process. The energy
generation or proton motive force may be altered
through mutation
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3. Efflux /influx mechanism Influx/efflux contd.

• Bacterial cells have an intrinsic capacity to restrict the entry
of small molecules especially gram neg-outer membrane is • When activated, the linker protein is believed to fold on
protective,gram pos no outer membrane hence more itself, bringing the AcrB and Tol C proteins in close contact,
antibiotic sensitive thus providing an exit path from the inside to the outside of
the cell.
• Restriction of influx is a physiological way to reduce toxixity
to bacterial cell. • Antibiotics are pumped out through this channel.
• The most wellstudied efflux system in E. coli is the
AcrAB/TolC system this system comprises of an inner
membrane proteinAcr B, and an outer membrane protein,
Tol C, linked by a periplasmic protein, Acr A
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4. Modification of target sites Altered cell wall precursor targets

• Alteration of ribosomal target sites-hence failure to inhibit • Glycopeptides like vancomycin-bind D-alanine-D-
protein synthesis and cell growth. alanine which is present at the termini of peptidoglycan
• Affected antibiotics are aminoglycosides, tetracylines,
macrolides, lincosamides. • The large glycopeptide molecules prevent the
incorporation of the pre cursors into the cell wall

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Alteration of target enzymes By pass inhibition

• Alteration of PBPs in Beta lactams • Development of auxotrophs-have growth factor
• SMX/TMP-production of a dihdropteroate requirements different from those of wild strain these
synthetase that is resistant to binding by mutants require subtrates that normally are
sulphonamides-plasmid mediated synthesized by the target enzymes and if these are
present in the environment the organisms grow
• Quinolones-DNA gyrase is made up of gyr A and gyr
despite inhibition by synthetic enzymes
B genes-mutations in gyr A result in resistance

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Factors promoting antimicrobial resistance

Antibiotic resistance  Exposure to sub-optimal levels of antimicrobial
 Exposure to microbes carrying resistance genes
 Inappropriate drug use-
Lack of quality control in manufacture or outdated antimicrobial
Injudicious use of antimicrobial agents
 Inadequate surveillance or defective susceptibility assays
 Poverty or war
 Use of antibiotics in foods-Antibiotics are used in animal feeds and
sprayed on plants to prevent infection and promote growth -Multi
drug-resistant Salmonella typhi has been found in 4 states in 18
people who ate beef fed antibiotics
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How to address the problem of increasing

antimicrobial resistance?
• WHO Antimicrobial resistance fact sheet 2016
• Antimicrobial resistance is a complex problem
Measures to control • Affects all of the society
• Driven by many interconnected factors
antimicrobial resistance • Single, isolated interventions have limited impact
• Coordinated action is required
• All countries need national action plans on AMR
• Greater innovation and investment are required in research
and development of new antimicrobial medicines, vaccines
and diagnostic tools.
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Clinical specimens

Methods of detecting
antimicrobial resistance
Wound swab, Blood,
Sputum, Eye swab, Ear
swab, Urine, Others

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Conventional Laboratory methods Antimicrobial Susceptibility

• Microorganisms, particularly bacteria, are tested
-Disc diffusion methods in vitro to determine whether they are
- Broth dilution for MIC susceptible to antimicrobial agents.
- Agar dilution • Disk Diffusion method, diameter of zone of
- E-test inhibition; Minimum Inhibitory Concentration
- Automated methods method. MICs are performed by CLSI-approved
for ID and MIC broth micro dilution
• These methods provides qualitative (S,I,R) of how
organisms may respond to drug in vivo &
quantitative results
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Agar Diffusion (Kirby-Bauer Disk) E-TEST METHOD (PDM

• Paper disk containing antibiotic is placed on lawn
of bacteria. Diameter of zone of inhibition is The E-Test is a novel method
inversely correlated with MIC. developed by AB Biodisk, Solna,
• Method has been standardized for commonly Sweden for direct quantification
isolated, rapidly growing organisms. of antimicrobial susceptibility
using an agar diffusion method.
The E-Test comprises a plastic
strip 50 x 5 mm predefined with
antibiotics on one side

Susceptibility Testing Methods

E-TEST METHOD (PDM Epsilometer) contd.
1). Agar Dilution: Incorporation of agent in
standardized media
2.) Agar Diffusion: Disk diffusion (Kirby-
Bauer), E-Test
The MIC is read where the zone of inhibition intercepts the 3) Broth Dilution: - Macro & Micro dilution
strip. Advantage: Speed of preparation, allows sensitivity 4) Automated methods or Systems e.g.
testing of single isolates, including fastidious species.
Increasingly used if susceptibility is required for critically ill
Vitek®, Phoenix®, MicroScan systems
patients e.g. meningitis, septicemia, anaerobic infections etc.


AST Methods Contd. Broth Dilution Method:

Quantitative results are reported as:
104 cfu
Minimum Inhibitory Concentration (MIC)
* most basic measurement of
antimicrobial activity against a target
* lowest antimicrobial concentration that
will inhibit growth of a test organism over
a defined range related to the organism mg/ml 4 2 1 0.5 0.25 0.125 0

growth rate Following inoculation & incubation the tubes are visually examined for
turbidity. MIC by convention is interpreted as the concentration of the
antibiotic contained in the first tube in the series to inhibit visible growth.
The last tube has no antibiotic and serves control.

Macro Dilution Results

In a dilution test, the lowest
dilution that prevents turbidity
(cloudiness) is the MIC. In this
example, the
MIC is 8 μg/mL. Broth from
samples without turbidity can
be inoculated onto plates
lacking the antimicrobial drug.
The lowest dilution that kills
≥99.9% of the starting
inoculum is observed on the
plates is the MBC.
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Micro Dilution Results AGAR DILUTION METHOD

A microdilution tray can also be used to
determine MICs of multiple antimicrobial
drugs in a single
assay. In this example, the drug
concentrations increase from left to right and
the rows with clindamycin, penicillin,
and erythromycin have been indicated to the
left of the plate. For penicillin and
erythromycin, the lowest • Here, the MIC is defined as the lowest concentration,
concentrations that inhibited visible growth allowing the growth of max of 5 colonies on the
are indicated by red circles and were 0.06 inoculated spot after adequate incubation time (usually
μg/mL for penicillin and 8 μg/
mL for erythromycin. For clindamycin, visible
18hrs). Growth control (18), Antimicrobial concentratñ
bacterial growth was observed at every 16mg/l (7/18), at concentration of 4mg/l (5/18) strains
concentration up to 32 μg/mL grew.
and the MIC is interpreted as >32 μg/mL.
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Molecular detection methods

Multiplex PCR was used to detect the
mecA and nuc gene targets using yellow
growth on mannitol salt agar containing
6mg/liter oxacillin (MSO-6) as a source of
How to monitor
M 1 2 3 4
antimicrobial use in

Size marker

Trinidad isolate
healthcare facilities
880 bp
2 Positive Control
3 Negative Control

4 Blank (without DNA)

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Antibiogram Monitoring Abx Use in health care facilities -

Local actions
 Antibiotic guidelines
◦ Narrow-spectrum agents for empirical treatment (where
◦ Minimize the use of 3rd gen cephalosporins, clindamycin,
carbapenems, fluoroquinolones
◦ Give guidance on dosage, duration, for iv to oral switch.
 Daily review of drug prescriptions by Pharmacists
 Microbiology ward rounds / MDT
◦ Daily Critical care rounds
◦ Daily follow up of patients with positive blood cultures
◦ Weekly Haematology MDT
◦ Weekly CDT Ward Round
◦ Weekly Orthopaedic ward
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Example of Empiric Antibiotic Guideline Criteria for Use

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Monitoring Abx Use in health care facilities -

Pediatric Empiric Guidelines Local actions
 AMS Committee set up
 AMS Program initiation or set up
 Antimicrobial Stewardship: Definition - “Optimal selection, dosage,
and duration of antimicrobial treatment that results in the best
clinical outcome for the treatment or prevention of infection with
minimal toxicity to the patient and minimal impact on subsequent
 A multi-faceted approach to influence antimicrobial prescribing,
institution-wide to improve outcomes, prevent resistance and
minimize excessive cost
 IDSA Guidelines. Clin Infect Dis 2007;44:159
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 Includes 28 recommendations
AMS actions
regarding implementation of AMS
Program • Microbiology input in patients positive blood cultures and
◦ Preauthorisation of abx ◦ Monitoring and adjustment other significant results
prescription programs for aminoglycosides
◦ Implement programs to increase • Daily review of patients on Critical Care
◦ Prospective audit and feedback
◦ Facility specific clinical practice appropriate use of oral abx and • ?microbiology review of patients on meropenem
guidelines timely transition from IV to oral
abx • ?microbiology review of patients on Tazocin >7 days
◦ Antibiotic stop orders on
prescriptions ◦ Selective reporting of abx • Antibiotic reports
◦ Computerized clinical decision ◦ Development of anti biograms • Written consultation system for all doctors on Meditech
support ◦ Rapid viral testing for respiratory
◦ Suggest against antibiotic cycling pathogens
9/6/2017 ◦Resistance
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Core elements of Performance in Nursing Homes

AMS actions for clinicians LEADERSHIP COMMITMENT

• Review antibiotics on ward rounds ACCOUNTABILITY

• Review antibiotics (and document) within 72 hours DRUG EXPERTISE

• Stop unnecessary antibiotics ACTION
• Review culture results
• Narrow the spectrum if possible TRACKING

• Switch to oral antibiotics if clinically appropriate REPORTING

• Contact microbiology if any concerns
CDC Core Elements of Antibiotic Stewardship for Nursing Homes.
The Joint Commission. Proposed Standard for Antimicrobial Stewardship in AHC, CAH, HAP, NCC, and OBS.
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The 7 key areas for future action

 1. Improving infection prevention and control
 2. Optimising prescribing practice
 3 Improving professional education, training and
public engagement
 4 Developing new drugs, treatments and
 5 Better access to and use of surveillance data
 6 Better identification and prioritisation of AMR "My child has an eating disorder...
research needs
She like vegetables"
 7 Strengthened international collaboration
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