Livret QA AST Bacterial Resistance

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Questions & Answers

onAntibioticSusceptibility Testing

and Bacterial Resistance


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QUESTIONS

ANDANSWERS

on SusceptibilityTesting

This guide is aimed at giving succinct answers to the

questions often asked about antibiotic susceptibility

tests and antibiotic therapy.

This guide was compiled with the help of Professor

Claude James SOUSSY, Service de Bactriologie,

Virologie et d'Hygine, Hpital H. Mondor, Crteil,

FRANCE and Doctor Emmanuelle CAMBAU, Service

de Bactriologie, Hpital Henri Mondor, Paris,

FRANCE.

PREFACE

Of all the laboratory examinations performed daily by

clinical microbiologists, susceptibility testing is of

particular clinical importance for correctly adapting

individual antibiotic therapy, monitoring the evolution

of bacterial resistance, and updating empirical

therapeutic strategies.

The choice of antibiotics to be tested must be

carefully determined depending on the bacterial

species tested and their natural resistance, local

epidemiology of acquired resistances, the site of

infection and local therapeutic requirements. In

certain cases, activity equivalences for several

antibiotics in the same class means only one

representative molecule of this class need be tested.

The intrinsic biological variation of bacterial

susceptibility to antibiotics constitutes the absolute

precision limit of susceptibility testing. Technical

factors must be controlled by rigorous

standardization of all the analysis stages (purity and

density of the bacterial inoculum, medium

composition, reagents, incubation conditions,

reading method and biological and clinical criteria

for interpretation of these results). Detailed and

continously updated national recommendations are

1 2


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available such as those compiled by the United

States Clinical and Laboratory Standards Institute

(CLSI) or Comit de l'Antibiogramme de la Socit

Franaise de Microbiologie (CA-SFM)*. Evaluation

procedures for analytical accuracy and precision

must also be applied regularly in order to guarantee

the quality assurance of the susceptibility test.

Progress still needs to be made in this field.

Rapid evolution of acquired resistance mechanisms

by clinically significant bacteria and the sometimes

weak expression of these resistance characteristics

justify the analysis of the "resistance phenotype" and

the possible use of additional tests. The aim is to

avoid categorizing bacteria as susceptible when they

express low level resistance in vitrobut are likely to

cause therapeutic failure. "Interpretive reading" can

be enhanced by the use of expert systems.

Finally, not only is the susceptibility test of immediate

interest for the clinician as a therapeutic adaptation

guide, but it also plays a role as an epidemiological

surveillance tool for local bacterial resistances. This

role requires periodic statistical analysis of the

cumulated levels of resistance per species, type of

specimen, and patient, in order to adapt the initial

empiric choice of antibiotic therapy while awaiting

laboratory test results. Finally, more detailed

statistical analysis enables the detection of

epidemics, notably intra-hospital, caused by multi-

resistant bacteria, which justify an enquiry and

appropriate infection control measures.

This brochure clearly explains basic facts

concerning the relevance and procedures of

susceptibility testing. The didactic quality of its

contents should enable anyone to understand the

essential elements required to perform and clinically

use susceptibility testing as a tool for optimizing anti-

infectious therapy.

Professor Claude James SOUSSY

Service de Bactriologie, Virologie et dHygine

Hpital Henri Mondor - Crteil - FRANCE

Professor Marc STRUELENS

Service de Microbiologie, U.L.B - Hpital Erasme

Brussels - BELGIUM

* French Susceptibility Committee

3 4


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The first aim of susceptibility testing is to measure

the susceptibility of a bacterial strain, presumed to

cause an infection, to one or several antibiotics. In

effect, in vitrosusceptibility is a prerequisite for the

in vivo efficacy of an antibiotic therapy. The

susceptibility test serves first and foremost to orient

individual therapeutic decisions.

The second aim of the susceptibility test is to

monitor the evolution of bacterial resistance. It is

due to this epidemiological follow-up by ward,healthcare establishment, region or country that

empiric antibiotic therapy can be adapted, and

antibiotic clinical spectra regularly revised.

Moreover, the detection of a large number of patients

infected with multiresistant bacterial strains at one

time and in the same place can influence some

healthcare decisions, such as the implementation ofprevention programs in hospitals.

Dual interest :

Individual (to administer, check and

sometimes modify therapy) and

Epidemiological

5

Why performsusceptibility testing ?

1.

6

Evolution of bacterial resistances - Community-acquired

Evolution of bacterial resistances - Hospital-acquired

E. coli/ ampicillin

S. aureus/ oxacillin

P. aeruginosa/fluoroquinolones

E. cloacae/3rdgen.cephalosporins

K. pneumoniae/3rd gen.cephalosporins

Enterococci /vancomycin

40

30

20

10

0

% of resistant strains

50

1975 1985 1998 2006

90

70

40

30

20

10

0

80

1975 1985 1998 2006

% of resistant strains

50

S. aureus/penicillin G

E. coli/ ampicillin

S. pneumoniae/

penicillin GS. pneumoniae/erythromycin

H. influenzae/ampicillin

Streptococci A /penicillin G


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Each time bacteria considered to be responsible for

an infection are isolated from a bacteriological

specimen.

Establishing the need for susceptibility testing

requires a close working relationship between

microbiologists and clinicians.

Sometimes microbiologists cannot definitely

determine if susceptibility testing is required, without

obtaining the clinical information that only a clinician

can provide.

For example, a commensal bacterium can cause an

infection in an immunocompromised patient or at a

specific body site.

Clinical symptoms can also be a determining factor

when deciding whether to perform susceptibility

tests (e.g. diagnosis of urinary tract infection with alow bacterial count).

In return, the information provided by susceptibility

tests will be useful to the clinician, not only for

prescribing an adapted antibiotic therapy to the

patient, but also for modulating the empiric antibiotic

therapy. This issue will be further examined in the

following questions.

Need for close working relationship between

microbiologists and clinicians.

7

When should a susceptibility testbe performed ?

2. Can susceptibilityand/or resistance of bacteriato an antibiotic be predicted ?

3.

8

Each antibiotic is characterized by a natural

spectrum of antibacterial activity. This spectrum is

the list of bacterial species which, in their "nave"

state, have their growth inhibited by theconcentration of antibiotic susceptible to work in

vivo. These bacterial species are said to be naturally

susceptible to this antibiotic. Bacterial species

which are not listed in this spectrum are said to be

naturally resistant.

Natural resistance is a stable characteristic ofall strains of the same bacterial species.

Knowledge of natural resistances enables the

inactivity of a molecule to be predicted in

relation to the identified (after collection) or

probable (in cases of empiric antibiotic therapy)

bacteria. It sometimes constitutes an

identification aid since some species can becharacterized by their natural resistance.

Examples :

Natural resistance of Proteus mirabilisto tetracyclines and

colistin.

Natural resistance of Klebsiella pneumoniae to

aminopenicillins (ampicillin, amoxicillin).


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Antibiotics Bacteria

Aminoglycosides Streptococci

Anaerobes

Macrolides Enterobacteriaceae

Glycopeptides Gram-negative bacteria

Colistin Gram-positive bacteria

Examples of natural resistance

Antibiotics

Penicillin G

Aminopenicillin

Aminopenicillin+ -lactamase

inhibitor

C1G

Cephalosporins C2G

C3G

Nalidixic acid

E. coli

Salmonella

Enterobacteriaceae

KlebsiellaEnterobacter

Serratia

*

Acquired resistance is a characteristic specific to

some strains, within a naturally susceptible

bacterial species, in which the genotype has been

modified by gene mutation or gene acquisition.

Contrary to natural resistances, acquired

resistances are evolutive and their frequency isoften dependent on the use of antibiotics. Given

the evolution of acquired resistances, the natural

activity spectrum is no longer sufficient to orient

the choice of antibiotic therapy for numerous

10

Pseudomonas

aeruginosaStreptococci Enterococci Staphylococci

= Natural resistance

* variable depending on the moleculeand species

C1G : 1st generation cephalosporins

C2G : 2nd generation cephalosporins

C3G : 3rd generation cephalosporins

Low levelresistance

bacterial species. Susceptibility testingtherefore

becomes essential.

Natural resistance : permanent

characteristic of the species,

which is known and predictable.Acquired resistance : characteristic

of some bacterial strains, which is

evolutive, unpredictable and justifies

the need for susceptibility testing.


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Example : erythromycin

Note : other pathogens, e.g. atypical bacteria such as Chlamydiae,

where clinical efficacy has been shown are mentioned separately.

Clinical spectrum of activity :

- useful for empiric antibiotic therapy

- depends on the frequency of the resistance

and the in vivoactivity of the antibiotic

The measurement of the susceptibility of a bacterium

to an antibiotic is based on the determination of the

Minimum Inhibitory Concentration (MIC).

The MIC is defined as the lowest concentration of a

range of antibiotic dilutions which inhibits all visible

bacterial growth.

It is the fundamental reference value which enables

a range of antibiotic activity to be established for

different bacterial species.

Various laboratory techniques enable the MIC value

to be measured or estimated semi-quantitatively in

routine use :

13

How is the bacterial susceptibilityto an antibiotic measured ?

5.

Susceptible European range of acquiredresistance(percentages to be insertedonly when appropriate as indicatedin the text above)

S.pyogenes 2 - 40%

S.pneumoniae 0 - 40%

Intermediate

H.influenzae

Resistant

Enterobacteriaceae

14

Principle

Bacterialgrowthmeasuredaccording to anantibioticconcentrationgradient

18 hrs

Timeto results

MANUALMETHOD

S

SEMI-AUTOMATED

ORAUTOMATED

METHODS

ATB strip

ATBrapid ATB

Agar diffusion

VITEK

VITEK 2 cards

Microtiter plate

Microtiter plate

Bacterialgrowthmeasuredaccordingto 2 or severalantibioticconcentrations

18 hrs

Bacterialgrowthmeasuredaccording toone (4 hrs)or 2 antibioticconcentrations

(18 hrs)

4 hrs to18 hrs

Kinetic analysisof bacterialgrowth

4 hrs to18 hrs


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For a given antibiotic, according to the NCCLS (M 100

-S15 January 2005), a bacterial strain is S, I, R as

follows (they are also similar to those of the CA-SFM1):

These different routine methods enable a bacterial

strain to be categorized according to its

susceptibility for the antibiotic being tested. This

strain is said to be Susceptible (S), Intermediate (I)

or Resistant (R)to the antibiotic.

Two antibiotic concentrations, known as

"breakpoints" determine these three categories :S, I, R.

These concentrations are set by National Expert

Committees, such as the United States Clinical and

Laboratory Standards Institute (CLSI) or Comit de

l'Antibiogramme de la Socit Franaise de

Microbiologie (CA-SFM), using bacteriological,

pharmacokinetic and clinical criteria. They areregularly revised.

Therapeutic categories S, I, R :

expression of susceptibility test results

for a bacterial strain

15

What do the categoriesS, I, R mean ?

6.

16

Susceptible (S)

The "susceptible" category implies that an

infection due to the strain may be appropriately

treated with the dosage of antimicrobial agent

recommended for that type of infection and

infecting species, unless otherwise

contraindicated.

Intermediate (I)

The "intermediate" category includes isolates

with antimicrobial agent MICs that approach

usually attainable blood and tissue levels and for

which response rates may be lower than for

susceptible isolates. The "intermediate" category

implies clinical applicability in body sites where

the drugs are physiologically concentrated (e.g.

quinolones and-lactams in urine) or when a high

dosage of a drug can be used (e.g. -lactams).

The "intermediate" category also includes a

"buffer zone" which should prevent small,

uncontrolled technical factors from causing major

discrepancies in interpretations, especially for

drugs with narrow pharmocotoxicity margins.

Resistant (R)

Resistant strains are not inhibited by the usually

achievable systemic concentrations of the agent

with normal dosage schedules and/or fall in the

range where specific microbial resistancemechanisms are likely (e.g. -lactamases) and

clinical efficacy has not been reliable in

treatment studies.


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Equivalence is the prediction of antibiotic in vivo

activity based on results obtained by testing another,

related antibiotic.

Example :

Equivalence between cephalothin which is tested and other

1stgeneration cephalosporins which are not tested. The result for the

other molecules can be equated with that obtained for cephalothin.

It is possible to test a restricted

number of antibiotics without limiting

therapeutic possibilities.

Antibiotics do not induce resistance, but select

resistant bacteria by eliminating susceptible

bacteria.

This is known as selective pressure.

The increase in the frequency of resistant strains is

most often linked to the intensive use of a specific

antibiotic.

19

Can antibiotics "induce"resistance ?

8.

Which methods enable resistancemechanisms to be demonstratedin vitro ?

9.

20

What is antibiotic equivalence ?

10.

For the moment, only specific techniques enable the

direct detection of biochemical mechanisms

(example : detection of -lactamase by hydrolysis of

nitrocefin) or genetic determinants of resistance(example : detection of the mecA gene responsible

for staphylococcal resistance to oxacillin). Some of

these techniques require molecular biology based

methods and are still not all used in routine.

Susceptibility test results can suggestthe presence

of a resistance mechanism.

2 types of genetic modifications

4 types of biochemical mechanisms

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; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ;

y y y y y y y y y y y y y y y y y















RNA

PBP

DNA

Main constituents of Gram-negative bacteria


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Some resistance mechanisms are weakly expressed

in vitro, although they are coded in the bacterial DNA.

Their expression in the human body, where conditions

are different, exposes the patient to the risk oftherapeutic failure. To avoid this, susceptibility testing

must be interpreted globallyto discern even a weakly

expressed resistance mechanism (by comparing

results for each antibiotic).

Therefore, due to the interpretation, a strain appearing

as falsely susceptible will be categorized as I or R.

23

Why is it necessary to interpretsusceptibility test results ?

13.

24

Example :

A strain of Klebsiella pneumoniaeproducing ESBL can appear in

vitroas being susceptible to 3rd generation cephalosporins. The

Susceptible result must be corrected to Intermediate or Resistant

since the use of this group of antibiotics can cause therapeutic

failure.

Through the judicious choice of antibiotics

tested, the interpretation of susceptibility

test results can help detect weakly

expressed resistance.

Resistance phenotypeobserved in vitro

Additionaltests

if required

Knowledge

of resistancemechanisms

Interpretive procedureInterpretive procedure

Resistance phenotypeobserved in vitro

Additionaltests

if required

Knowledge

of resistancemechanisms

Determinationof probableresistancemechanism

Validation/Corrections

Resultgiven

Validation/Corrections

Resultgiven

Determinationof probableresistancemechanism


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Validation of a susceptibility test result requires a

comprehensive knowledge of resistance mechanisms

and antibiotic activity. An Expert system is a software

package designed to help make decisions which, byintegrating this knowledge, automatically interprets

susceptibility tests, checks results and suggests the

necessary corrections. The Expert system contributes

to the reliability of the result given by :

ensuring consistency between the susceptibility

test result and bacterial identification.

Example : Klebsiella pneumoniae - ampicillin S = improbablephenotype.

identifying improbable or impossible resistance

phenotypes.

Example : E. coli - cephalothin S - cefotaxime R = improbable

phenotype.

What is the role of an Expert system ?

14.

detecting insufficiently expressed resistances.

Example :

Detection of an extended spectrum -lactamase (ESBL) and

correction of S results to I or R for -lactams except for

cephamycins (example : cefoxitin) and imipenem.

indicating a rare phenotype in a given context.

The regular update of information constituting the

knowledge-base is essential. In effect, some

notions which are true at a given moment and for

a given place can be different at another time and

in another country.

Examples :

- MRSA strains have been resistant to gentamicin for a very long

time, but this association is statistically less true today.- Enterococci are frequently vancomycin-resistant in the USA but

are still rarely resistant in Europe.

Expert System : a tool for interpretation

of susceptibility test results.

The Expert SystemThe Expert System

Fact-base

Knowledge-base

InferenceEngine

InferenceEngine

Knowledge-base

Fact-base

Result checkedand corrected

Deductionof probableresistance

mechanism

Deductionof probableresistance

mechanism

Result checkedand corrected

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Some current resistance problems

15.

Are bacteria responsible for community-

acquired urinary tract infections affected

by antibiotic resistance ?

Although bacterial resistance is more frequent in

hospitals than in the community, bacteria that are

most often found in community-acquired

pathologies, such as E. coli, can acquire antibiotic

resistance.

For example, in 2005 the level of acquired resistance of E.colito

antibiotics frequently used for the treatment of urinary tractinfections were :

Europe US

Ampicillin 60% 54%

Amoxicillin/Clavulanic acid 30% 23%

Fluoroquinolones 24% 25%

Cotrimoxazole 60% 60%

What is the probability of infection by

Enterobacteriaceaewith extended spectrum

-lactamase (ESBL) or by MRSA strains ?

In patients from the community, the frequency of this

type of multiresistant bacteria (ie. with acquired

resistance to numerous antibiotics) is directly linked

to a previous hospital stay.

These bacteria are mainly found in hospitals, where

their multiresistance gives them a selective

advantage.

Generally transmitted from one patient to

another in the same healthcare unit (hospital,

clinic, nursing home, etc...), they are responsible for

nosocomial infections.

Active and rapid communication between the

microbiologist and the clinician in charge of the

patient, as well as the infection control team, enables

the necessary measures to be implemented to avoid

the spread of this type of bacteria (e.g. isolation of

the patient, reinforcement of hygiene rules, etc...)

Why is it now essential to check the

susceptibility of S. pneumoniaeto antibiotics

and notably to penicillin G ?

For over 10 years, pneumococcal resistance to

penicillin G has continuously increased in many

countries (less than 1 % in 1985, 10-50 % in 1998). This

resistance is generally associated with resistance to

other antibiotics (tetracyclines, macrolides,

cotrimoxazole...). This evolution questions empirical

antibiotic therapy for ENT (Ear, Nose and Throat),

bronchopulmonary infections, as well as meningitis

which are often caused by S. pneumoniae.

Resistance of S. pneumoniae to penicillin G is

extended to all -lactams, with different levels of

resistance depending however on the molecules of

this family. This requires further testing to be

performed (e.g. exact determination of the MIC for

penicillin G, amoxicillin, cefotaxime...).

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1. 1996 Report of the Comit de l'Antibiogramme de laSocit Franaise de Microbiologie.Clinical Microbiology and Infection 1996; 2 (suppl, 1) 51 - 549

2. National Committee for Clinical Laboratory StandardsPerformance Standards for Antimicrobial Disk

Susceptibility Tests - Sixth Edition; M2-A6, 19993. Courvalin P., Goldstein F., Philippon A., Sirot J.

L'antibiogramme, 1re Ed. Paris : MPC Vigot, 1985

4. Courvalin P., Flandrois J.P., Goldstein F., Philippon A.,Quentin C., Sirot J.L'antibiogramme automatis, 1re Ed. Paris : MPC Vigot,1988

5. Goldstein F., Soussy C.J., Thabaut A.Le spectre antibactrien d'un antibiotique.Actualits mthodologiques : biostatistique, aspects

mthodologiques de l'valuation des anti-infectieux.Springer-Verlag, France 1993

6. Hanberger H., Garcia-Rodriguez J-A., Gobernado M.,Goossens H., Nilsson LE., Struelens MJ., and the Frenchand Portuguese ICU Study Groups.Antibiotic susceptibility among aerobic Gram-negativebacilli in intensive care units in 5 European countries.JAMA 1999 ; 281 : 67-71

7. Michael A.Pfaller, Ronald N.Jones, Gary V.Doern,Kari Kugler, and the SENTRY participants group.

Bacterial Pathogens Isolated from Patients withBloodstream Infection : Frequencies of Occurrence andAntimicrobial Susceptibility patterns from the SENTRYAntimicrobial Surveillance Program (United States andCanada, 1997)Antimicrobial Agents and Chemotherapy, 1998 ; 42 :1762-1770

8. M.A.Pfaller, R.N.Jones, G.V.Doern, H.S.Sader, K.C.Kugler,M.L.Beach, and the SENTRY participants group.Survey of Blood Stream infections Attributable

to Gram-Positive Cocci : Frequency of Occurrenceand Antimicrobial Susceptibility of Isolates Collected in1997 in the United States, Canada, and Latin America from

the SENTRY Antimicrobial Surveillance Program.Diagnostic microbiology and infectious disease 1999 ; 33 :283-29

BIBLIOGRAPHY

CONCLUSION

Antibiotic susceptibility testing, at the interface

between the clinical diagnosis and the therapeutic

decision, is a key element essential for guiding both

microbiologically-documented and empiric antibiotic

therapy.

The evolution of bacterial resistance, as well as the

development of new antibiotics and laboratory

techniques make a close working relationship

between the microbiologist and the clinician more

necessary now than ever before.

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Livret QA AST Bacterial Resistance

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