INTERPRETATION OF

ANTIBIOGRAMS
Trends of Change
Dr.T.V.Rao MD
1

Dr.T.V.Rao MD

3/24/2016

The
2 Program file summarises on New Trends of Change
in Antibiotic sensitivity and resistance changes
Problem with Inconsistent on
phenotype

Implications Good reminder of

intrinsic and exceptional
resistance
Everything about Microbes
changes faster than we think
limited science
Dr.T.V.Rao MD

3/24/2016

The purpose of Antibiograms

The purpose of a
susceptibility test is to in
vitro determine the
sensitivity or resistance of
pathogenic aerobic and
facultative anaerobic
bacteria to various
antimicrobial
compounds.
Dr.T.V.Rao MD

3/24/2016

Requirements to create Antibiograms

Dr.T.V.Rao MD

The use of standardized

culture media and careful
control of all test
conditions are
fundamental
requirements in the
microbiological assay of
antibiotics in order to
achieve satisfactory test
results.
3/24/2016

Monitoring of Antibiograms is crucial in Hospital

practice

It is crucial to monitor
emerging trends in
resistance at the local level
to support clinical decision
making, infection-control
interventions, and
antimicrobial-resistance
containment strategies
Dr.T.V.Rao MD

3/24/2016

Various methods of antibiotic susceptibility

testing are:
1. Quantitative
Methods
2. Qualitative Methods
3. Automated
Susceptibility Tests
4. Newer NonAutomated
Susceptibility Tests
5. Molecular
Techniques
Dr.T.V.Rao MD

3/24/2016

Quantitative Methods

In these tests, the minimum amount of antibiotic that

inhibits the visible growth of an isolate or minimum
inhibitory
concentration (MIC) is determined. Bacterial isolate is
subjected to various dilutions of antibiotics. The
highest dilution of antibiotic that has inhibited the
growth of bacteria is considered as MIC. These tests
can be performed on broth or agar.
Dr.T.V.Rao MD

3/24/2016

Quantitative Methods

1. Broth dilution methods

a. Macro broth dilution
MIC tests
b. Micro broth dilution
MIC tests
2. Agar dilution methods
Dr.T.V.Rao MD

3/24/2016

AST standards

MIC taken as standard

measure of
susceptibility
Agar dilution
Broth dilution
Micro broth dilution
Dr.T.V.Rao MD

3/24/2016

AST standards

10

MIC taken as standard measure of

susceptibility
Agar dilution
Broth dilution
Micro broth dilution

S/I/R Breakpoints defined by

BSAC/EUCAST/CLSI
Dr.T.V.Rao MD

3/24/2016

ISO standard

11

Broth microdilution
Mueller-Hinton
Broth
Inoculum 5x105
cfu/mL
Dr.T.V.Rao MD

3/24/2016

12

The rationale for automated systems

More reliable AST results

?closer to standard

Reduced scope for user error

Reproducible

Improved ID linked to AST

Embedded expert rules
Dr.T.V.Rao
3/24/2016
MD

13

What is a Antibiogram

Antibiograms are
collection of
information obtained
from Culture and
Sensitivity performed in
the institution within a
given time frame
Dr.T.V.Rao MD

3/24/2016

14

Dr.T.V.Rao MD

3/24/2016

15
Why
things go wrong in Antibiotic Sensitivity
testing

Excessive pre-incubation before discs

applied

Excessive pre-diffusion before plates

incubated
Incorrect incubation temperature (350C
for cefoxitin vs Staphylococci)

Incorrect incubation atmosphere (false

metronidazole)
Prepared by Trevor Winstanley for the BSAC
recommendations
Dr.T.V.Rao MD

3/24/2016

16 things go wrong in Antibiotic Sensitivity testing

Why

Incorrect incubation time

(VanB resistance in enterococci)
Inadequate illumination of plates when
reading
Incorrect reading of zone edges
(cefoxitin for the detection of
MRSAs)Incorrect template (wrong agent
or wrong version of the guidelines)
Prepared by Trevor Winstanley for the BSAC
recommendations
Dr.T.V.Rao MD

3/24/2016

We work with The ClSI

M39-A2 Guideline or Newer updates

17

CLSI M39-A2 is intended for

those involved in the preparation
and use of cumulative
antibiogram reports, as well as for
information technology
managers who are responsible
for designing and supporting the
clinical laboratory's data
management needs
Dr.T.V.Rao MD

3/24/2016

18

What the Document Contains

The document contains specific recommendations

for the collection, storage, analysis, and presentation
of data and includes sample templates that highlight
the recommendations. Critical issues addressed
include the recommended frequency of reporting,
the number of isolates to include in a statistic, and a
mechanism for eliminating multiple isolates of a given
bacterial species obtained from an individual patient
Dr.T.V.Rao MD

3/24/2016

19

Provides Information on Resistance and

Sensitivity of the tested isolate
Provides the % of
samples for a
given organism
which were
sensitive or
resistant to certain
antibiotics
Dr.T.V.Rao MD

3/24/2016

20

Potential sources of error in disc diffusion

antimicrobial susceptibility testing:
antimicrobial discs

Wrong agent or content used

Labile agent possibly deteriorated

Light sensitive agent left in light
Incorrect storage leading to deterioration
Disc containers opened before reachingroom temperature
Incorrect labelling of disc dispensers

Expiry date exceeded

Prepared by Trevor Winstanley for the BSAC recommendations
Dr.T.V.Rao MD

3/24/2016

Potential sources of error in disc diffusion

antimicrobial susceptibility testing: control strains

21

Contamination
Mutation
Incorrect inoculum density
Uneven inoculation
Old culture used not properly maintained
Prepared by Trevor Winstanley for the BSAC recommendations
Dr.T.V.Rao MD

3/24/2016

22
Antibiograms
are Important to Clinicians to
make decisions

An antibiogram is an
essential tool for any
clinician when treated an
infection empirically
Antibiogram can serve
as an alternative to a C&S
report until the results of a
C&S are available
Dr.T.V.Rao MD

3/24/2016

23

Antibiograms are Important to

Clinicians to make decisions

Dr.T.V.Rao MD

Antibiogram can
serve as an
alternative to a
C&S report if no
organism is grown
out of a C&S
despite high
clinical suspicion
of an infection

3/24/2016

24

Dr.T.V.Rao MD

3/24/2016

25

Disk diffusion test

In this method the standardized bacterial isolate is

spread on an agar plate and then paper disc
containing specific concentration of antibiotics are
placed and incubated at 37oC overnight. If the
isolate is susceptible to the antibiotic, it does not
grow around the disk thus forming a zone of
inhibition. Strains resistant to an antibiotic grow up to
the margin of disk. The diameter of zone of inhibition
must be measured and result read from the Kirby
Bauer chart as sensitive, intermediate or resistant
Dr.T.V.Rao MD

3/24/2016

Clinical utility

26

The greater the number of

isolates, the more
accurate the sensitivity
results for the given
organism The greater
the number of isolates, the
higher probability of
isolating the given
organism
Dr.T.V.Rao MD

3/24/2016

27

Parts of the Antibiogram % Susceptible

Percentage of
isolates of the
given organism
which is
susceptible
(sensitive) to the
given Antibiotic
Dr.T.V.Rao MD

3/24/2016

Resistance

28

Reflects the % of organism which are

resistant to certain Abx Clinical Utility:
Assists in determining if coverage for MDR
organisms in the empiric therapy are
necessary Information can be obtained
from the % susceptibility chart Important
for MRSA, VRE, ESBL, and KPC
Dr.T.V.Rao MD

3/24/2016

29

Antimicrobial agents to analyse.

Only results for antimicrobial agents that are

routinely tested and clinically useful should be
presented to clinicians. To avoid biases introduced
by selective reporting practices (e.g., reporting
broad-spectrum agents only for bacteria with
resistance to primary agents), the analysis database
should include the results for all antimicrobials
tested, including those agents that may not be
routinely reported to clinicians.
Dr.T.V.Rao MD

3/24/2016

Do30not do disc contain test with Vancomycin

for Staph aureus
Disc testing should NOT be used.
An MIC based method should be
used.
Whichever method is used, a control
strain

(ATCC 25923 or ATCC 29213) should be

tested in parallel with each run of test
organisms
Monitor performance of QC (even
within acceptable range
Dr.T.V.Rao MD

3/24/2016

31

Antimicrobial agents to analyse.

Results for antimicrobials

tested only against drugresistant strains as part of
reserve or second-line testing
panels are generally biased
towards higher rates of
antimicrobial resistance and
should not be considered to
be representative.
Dr.T.V.Rao MD

3/24/2016

32

Should be familiar with trends in Antibiotic

Science

Susceptibility testing requires

Reliable/reproducible test
Relationship between test and clinical outcome
B. cepacia
Testing poorly reliable/reproducible
Poor definition of relationship with outcome
S. maltophila
Testing poorly reliable/reproducible
Poor definition of relationship with outcome
Co-trimoxazole AST remains challenging
Dr.T.V.Rao MD

3/24/2016

33

Inherent resistance we mostly

agree on - non-fermenters

Pseudomonas, Acinetobacter - ampicillin, cephs

except ceftazidime, tetracycline, chloramphenicol,
ertapenem
S. maltophilia - all -lactams except ticar-clav
,aminoglycosides, trimethoprim alone

Burkholderia - aminoglycosides & colistin

Elizabethkingella Dr.T.V.Rao MD

-lactams except pip-tazo

3/24/2016

Exceptional
resistance

34

Enterobacteriaceae
Meropenem / imipenem
P. aeruginosa,
Acinetobacter colistin
Bacteroides
metronidazole &
carbapenems
Dr.T.V.Rao MD

3/24/2016

35

Disc testing unreliable Colistin

Colistin AST
Disc testing unreliable
MIC testing remains challenging
(method development)
Vancomycin AST for staphylococci
Disc testing unreliable
MIC testing remains challenging (BP
close to wild-type popln
Dr.T.V.Rao MD

3/24/2016

36

B. cepacia complex

Multiple resistance mechanisms

(?variable expression)
No reliable gold-standard test
No defined relationship between MIC &
clinical outcome
Wide MIC distribution across PKPD BP

No AST method
Dr.T.V.Rao MD

3/24/2016

37

B. cepacia complex:
a reliable/reproducible test?

Gold standard
MIC performed to ISO
20776-1 (2006)
Micro broth dilution
Mueller-Hinton Broth
Dr.T.V.Rao MD

3/24/2016

38

Stenotrophomonas maltophilia

Multiple resistance mechanisms

(?variable expression)
No gold standard test
Results markedly affected by

incubation temperature, culture

medium and technique
V little data to relate test to clinical

outcome
Dr.T.V.Rao MD

3/24/2016

All the Microbiologists should be familiar with

39

For the seriously-ill patient, fast

microbiology is more valuable
than precise microbiology
And, mostly, microbiology
moves at the pace
it did in Flemings time: 48h
from specimen to susceptibility
result
Dr.T.V.Rao MD

3/24/2016

40

The truth about our Microbiology

Departments

Far more potential to

accelerate seeking
resistance genes than
phenotypes
It means we detect
many matters later than
the Bactria have evolved
Dr.T.V.Rao MD

3/24/2016

41

Does the absolute truth exists in

Antibiotic Sensitivity testing

Why we think EUCAST (& CLSI) ESBL advice is wrong

Pharmacokinetics more variable than we admit
Outcomes are inconsistent vs. low-MIC ESBL
&carbapenemase producers
Inoculum effect (TW)
Susceptibility tests less precise than we wish
Mechanism detection will become faster than susceptibility
testing
Ref Livermore et al. JAC 2012;67:1569
Dr.T.V.Rao MD

3/24/2016

42

The rationale for automated systems

More reliable AST results

Improved ID linked to AST
Embedded expert rules
Improved speed of results
Increased number of agents
tested
Dr.T.V.Rao MD
Automated
systems: an overview

3/24/2016

The
rationale
for
automated
systems
43
More reliable AST results

Improved ID linked to AST

Embedded expert rules

Improved speed of results

Increased number of agents
tested

Laboratory workflow
Staff skill mix
Dr.T.V.Rao MD
Automated
systems: an overview

Epidemiological software

3/24/2016

44

Available systems

Micros can Walkaway

Biomerieux Vitek 2

BD Phoenix
Dr.T.V.Rao MD

3/24/2016

Micro Scan

45

96 well plates
AST based on
conventional micro broth
dilution MIC
ID currently biochemical
Dr.T.V.Rao MD

3/24/2016

46
Conventional panels

Micro scan

Enterobacteriaceae, gram-negative non-fermenters,

staphylococci, enterococci
Combo, MIC-only, ID-only

Rapid panels
2 - 2.5 hour ID (pre-formed enzymes)
AST for Gram negative organisms (MIC > 4 hrs)

Synergies plus panels

2 - 2.5 hour ID (pre-formed enzymes)
Broth microdilution Read-when-ready MIC
Resistance detection flagged in as few as 4.5 hours
All results finalized in 16/18 hours

Additional

Panels read by

WalkAway systems
autoSCAN-4
Manual

Yeast ID, Anaerobe ID, HNID

MICroSTREP plus - dry format
ESL plus -ESL Confirmatory Panel
Dr.T.V.Rao MD

3/24/2016

Vitek 2

47

AST based on broth

susceptibility with specific
cards
ID currently biochemical
Growth/reactions
continuously monitored
Dr.T.V.Rao MD

3/24/2016

Vitek 2

48

ID Cards available for

Gram positives
Enterobacteriaceae
Non-Enterobacteriaceae
Neisseria/Haemophilus etc
Anaerobes
Yeasts
AST Cards available for
Gram positive cocci
Gram negative bacilli
Yeasts
Dr.T.V.Rao MD

Up to 22
antibiotics tested
on each card
3/24/2016

49

VITEK 2 MIC Interpretation

Interpretation of Raw Data
Growth Parameters

MIC
Raw Data
% Change
Slope
Area
SLPAR
0

10 11 12 13 14 15 16 17 18

TIME (hours)

Dr.T.V.Rao MD

3/24/2016

Vitek 2

50

Expert rules within

the Advanced
Expert System
(AES)
Extensive
Knowledge Base
>20,000 MIC
distributions
>2,500 Phenotypes
>200 Resistance
Mechanisms
Dr.T.V.Rao MD

3/24/2016

Vitek 2

51

CARD Type

Time to Result

GN ID
GP ID

2 to 10 hours
2 to 8 hours

NH ID

6 hours

ANC ID
YST ID
GN AST
GP AST
AST-YS01

6 hours
18 hours
2 to 18 Hours
2 to 18 Hours
6 hours

Dr.T.V.Rao MD

3/24/2016

Phoenix

52

AST based on broth

susceptibility with specific
cards
ID currently biochemical
Growth/reactions
continuously monitored
Dr.T.V.Rao MD

3/24/2016

Phoenix

53

AST only or Combo panels available

for

GN (Enterobacteriaceae/
Nonfermenters)
GP (Staphylococci/
Enterococci/
Streptococci)
Streptococci ID/AST
Dr.T.V.Rao MD

3/24/2016

Phoenix

54

Readings for each well taken and calculated every 20

minutes
ID uses red, green, blue and UV light sources

AST measures turbidity and color change

Each well divided into pixels
Readings taken for each pixel
Entire process takes 7 minutes

Pixel data analyzed for each well

Readings for each well compared to previous reading and
baseline
Well is complete when specific amount of change is not
seen
Substrate/antimicrobic algorithm dependent
Instrument will post results when enough data collected
Dr.T.V.Rao MD

3/24/2016

Phoenix

55

Expert rules within

BDXpert based on
CLSI/SFM/DIN/EUCAST
rules bases

Dr.T.V.Rao MD

3/24/2016

56

Comparison of systems
Microscan

ID available

AST available
Reading
MIC
Expert system
Biochemical ID
Plans for integration with nonbiochemical ID systems
Dr.T.V.Rao MD
Epidemiological software

GPOS
GNEG NH
AnO2
Yeast
GPOS
GNEG

Vitek 2
GPOS
GNEG
NH
AnO2
Yeast
GPOS
GNEG
Yeast

Phoenix
GPOS
GNEG
GPOS
GNEG

Continuous automated
or manual
Y
Published rules

Continuous automated

Continuous automated

+/Phenotypic comparison

+/Published rules

3/24/2016

Performance

57

ID
Correct to genus >95%
Correct to Species>90%
Difficulties with difficult organisms
Non-fermenters
Shigella etc

AST
Concordant with other methods >95%
Specific issues with specific drug-bug combinations & certain
instruments
VISA/VRSA

Beta-lactams & Pseudomonas

Dr.T.V.Rao MD

3/24/2016

Performance

58

ID/AST performance relies on

correct ID, correct AST plus
appropriate expert
interpretation
Incorrect ID can lead to incorrect
AST

Up to 5% errors mean that

results should be examined
critically
Dr.T.V.Rao MD

3/24/2016

Advantages of Automated systems in

59
AST
Automated systems can provide
Reliable AST integrated with ID & expert rules
Improved speed of results

Caution about staff skill mix and interpretation

of results
Microscan/ Vitek 2 /Phoenix have similar
performance overall
Results should be examined critically
Dr.T.V.Rao MD

3/24/2016

References

60

1Why some tests are no longer recommended Robin A Howe, Cardiff, UK

Public health wales
2ANTIBIOTIC SUSCEPTIBILITY TESTING by Dr Sridhar Rao PN Open web
resource
3Expert rules for Gram-negatives Trevor Winstanle Sheffield Teaching
Hospitals Presented on behalf of David Livermore University of East Anglia
& Health Protection Agency
4 Internal Quality Assurance Jenny Andrews Secretary of the BSAC
Working Party on Susceptibility Testing

5Automated systems: an overview Robin A Howe Cardiff Public health

wales
Dr.T.V.Rao MD

3/24/2016

Attention of Viewers

61

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3/24/2016

62

Program Created by Dr.T.V.Rao MD for Benefit

of Medical and Paramedical Professionals in the
Developing World Created from World Wide
Resources
Email
doctortvrao@gmail.com

Dr.T.V.Rao MD

3/24/2016