In vitro study of antibiotic

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I- lntroduction :

The purpose of performing an antibiogram is to predict the sensitivity of an organism to one or more antibiotics in an essentially optical therapeutic. It also serves :

  • In epidemiological surveillance of bacterial resistance ;
  • In bacterial identification by the identification of natural resistance.

Keep in mind that in practice, studying the effect of antibiotics in vitro and usually under standard culture conditions. So, it must be determined correlations to assume the in vivo efficacy of the antibiotic and thus to success (or failure) treatment on the biological in vitro database.

II- bacteriostasis :

1- Definition :

Bacteriostasis corresponds to a slowdown in the growth of a bacterial population, up to the cessation of growth. This only applies if the bacteria was in the growth phase before contact. Otherwise a lack of development may also correspond to a pronounced increase in latency.

Bacteriostasis can be studied in the liquid medium, for example by photometric monitoring of the growth of microorganisms in the presence of varying concentrations of antibiotics.

2- MIC = Minimum Inhibitory Concentration :

The most commonly used parameter to evaluate the effect of an antibiotic is the IJC. It corresponds to the minimum concentration of antibiotic that inhibits visible growth of the organism 24H. The CMI thus explores the bacteriostatic effect only, (note that its use is not justified in an immunocompromised patient).

Note good correlations biological-clinical jobs IJC, which is a good predictor of efficacy of antibiotic therapy : When it exceeds a certain value treatment failure is usual : when it is less than another value success is virtually assured. Between the two previous values, the prediction is impossible

3- MIC determination :

The IJC is not, to a given bacterium, biological constant. It is defined as the lowest concentration of a range of dilutions of antibiotic half half which leads to inhibition of any visible bacterial growth.

The method by successive dilution in solid medium is the reference method for bacterial susceptibility to antibiotics. This determination requires strict standardization of protocol experimental (influence Nnoculum, incubation, the seeding technique, antibiotics to be tested, reading, the culture medium and quality controls), any modification of experimental conditions makes interpretation difficult.

Other methods are applicable successive dilutions in liquid medium (bacterial growth assessed by the appearance of a disorder) ; E-Test (strips impregnated with a gradient of antibiotic).

III- Sensitivity Concept / Resistance :

1- Definition of resistance :

Bacterial resistance to antibiotics is either natural, is acquired.

The natural resistance of a species or genus is :

  • A feature, for all strains of the species or genus
  • Carried by a chromosome always transmitted to offspring : vertical transmission

A character to define the wild or sensitive phenotype of the species Acquired resistance, For its part :

  • Concerns only a more or less large proportion, variable in time
  • Resulting from genetic modification by mutation or acquisition of plasmids or transposons, (Extra chromosomal resistance) horizontally transmissible, sometimes between different species

Defines phenotypes "resistant".

Cross-resistance is expressed, they, within the same class of antibiotics and are due to the same mechanism of resistance.

2- Determination of class S / L / R :

the critical values ​​were determined which define the clinical categories and provide a guide for determining the susceptibility of bacteria to antibiotics. The critical values ​​defined for the concentrations and the diameters of zones of inhibition, as well as specific recommendations to certain species or groups of antibiotics

Three clinical categories were selected for the interpretation of sensitivity in vitro : Sensible (S), Resistant (R) and Intermediate (I).

The categorized S strains : are those for which the probability of therapeutic success is strong in the case of a systemic treatment with the recommended dose

The categorized R strains: are those for which there is a high probability of treatment failure regardless of the type of treatment and the dose of antibiotic used.

Stem categorized I : are those for which therapeutic success is unpredictable. These strains are a heterogeneous set for which the results obtained in vitro are not predictive of a therapeutic success.

3- Establishment of critical values ​​defining clinical categories :

The diffusion method using the relationship between MIC and diameter of inhibition around a source of antibiotic, reading is relatively straightforward. For bacteria and antibiotic given, the inhibition measured diameter is compared to the zone diameter :

  • < the lower critical diameter, the strain is classified R.
  • > u is critical diameter greater than D, the strain is classified S.

Critics diameters and D correspond respectively to high and low critical concentrations C c.

The concentration values ​​and critical diameters are defined for each antibiotic

4- Relationship with the CM I :

The eyes of concentrations and critical diameters are considered :

-► Sensitive (S) : strains for which the MIC of the antibiotic tested ^ is the low critical concentration (c), which is equivalent to a diameter > the critical diameter D

-► Resistant (R) : vis-à-vis which strains the MIC of the antibiotic tested, is > high critical concentration C, corresponding to a diameter < diameter critic

-► From intermediate susceptibility (I) : vis-à-vis which strains the MIC of the antibiotic tested and the corresponding diameter are between the two critical concentrations and two zone diameter.

Categories CMI (mg / L) Diameter (mm)
Sensible CMI ≤ c Diameter ≥ D
Resistant CMI > C Diameter < d
Intermediair c < CMI ≤ C d ≤ Diameter < D

IV- Concept of spectrum :

À chaque antibiotique est associée une liste d’espèces bactériennes qui constitue lespectre d’activitéde la molécule. Natural spectrum, established in early studies before any therapeutic use, remains stable by definition since it does not take into account the proportion of bacteria that have acquired antibiotic resistance after use. This proportion increases with time because the use of the antibiotic exerts selective pressure for the emergence of mutants or strains carrying extrachromosomal resistance factors. This concept must be known to the clinician because it explains the seemingly paradoxical situations : for example, natural spectrum of penicillin G comprises Staphylococcus aureus whereas currently 90% strains are resistant by producing penicillinase.

V- Methods for in vitro study :

A- Antibiogramme :

1- Principe :

L & rsquo; susceptibility testing is to determine the Minimum Inhibitory Concentration (CMI) a bacterial strain vis-à-vis various antibiotics.

2- conventional techniques :

dilution methods

Dilution methods are performed in liquid or solid medium in the middle. They include putting a standardized bacterial inoculum in contact of increasing concentrations of antibiotics in a geometric progression of ratio 2.

In liquid medium (figure 1), l & rsquo; bacterial inoculum is distributed in a series of tubes (method macrodilution) or cupules (microdilution method) containing the antibiotic. after incubation, the MIC is indicated by the tube or cup containing the lowest concentration of & rsquo; antibiotic where no growth n & rsquo; is visible.

Figure 1 : MIC determination by dilution in liquid medium.

Ex : The MIC of the tested strain is 2 pg / mL (first tube in which no growth n & rsquo; is visible to the & rsquo; naked eye).

In solid medium (figure Ibis), l & rsquo; antibiotic is incorporated into an agar medium poured in Petri dishes. The surface of the agar was seeded with an inoculum of strains to study. after incubation, MIC of each strain is determined by the & rsquo; inhibition of growth on the medium containing the lowest concentration of antibiotic. The middle agar dilution method, carried out with a range of concentrations in geometrical progression because of 2 is the reference method.

Figure 1bis : MIC determination by agar dilution.

Ex : The MIC of strain 3 vis-à-vis the & rsquo; antibiotic incorporated into the agar is Ipg / mL. The MIC of strain 2 is of 2 pg / mL. The MIC determinations stem 1 and 4 require testing stronger increasing concentrations of antibiotic.

Delivery methods : antibiogramme standard

Available methods or standards susceptibility are most used by diagnostic laboratories.

Discs of blotting paper, impregnated antibiotics to be tested, are deposited on the surface of & rsquo; an agar medium, previously inoculated with a pure culture of the strain to be studied. From the & rsquo; application of discs, antibiotics uniformly diffuse so that their concentrations are inversely proportional to the distance the disc. after incubation, CD s & rsquo; surround areas & rsquo; circular inhibition corresponding to an absence of culture. When the technique is well standardized, the diameters of the zones & rsquo; inhibition depend only on the germ sensitivity.

3- Standardisation :

The reliability of results & rsquo; antibiotic sensitivity is influenced by many parameters that must be strictly controlled. The standardization is governed by documents from the & rsquo; O.M.S. and various national committees. Depending on the country, there may be technical variations and it is important to follow the same procedure as for the & rsquo; establishment of standard curves.

4- Results :

Interpretative reading of the & rsquo; susceptibility

The interpretative reading of & rsquo; susceptibility is based on knowledge of resistance phenotypes. Its main goal to transform a result categorized “sensible” result in a “intermediate” or “resistant” due to a risk of treatment failure. The interpretive reading requires correct identification of the strain and a method & rsquo; perfectly standardized susceptibility testing. The identification of highly improbable resistance phénoypes given the & rsquo; strain identification should lead to check & rsquo; bacterial identification, to control the purity of the & rsquo; inoculum and control technique of & rsquo; susceptibility.

Currently, there are computerized systems & rsquo; helps validate the results : expert systems that allow for abnormal results. This validation check is to :

  • Check the consistency germ / susceptibility
  • Detecting impossible resistance phenotypes
  • Detect & rsquo; & rsquo absence; an associated resistance
  • To recognize abnormal phenotypes may correspond to new resistance methods.

Rq : Generalizing the results to untested antibiotic choice of tested antibiotics is primarily based on the & rsquo; identification of the organism and the knowledge of its natural resistance. This choice also depends on the site of the & rsquo; infection.

Among the antibiotics that could & rsquo; be used therapeutically, all molecules are not taken into account. Indeed, knowledge of the families & rsquo; antibiotics and cross-resistance mechanisms makes it possible to include in the & rsquo; antibiogram that & rsquo; a small number of representative molecules.

5- other technical :

agar Technique : the E-Test®

The precise determination of the CM by the reference method is difficult to use in daily practice. Marketing d & rsquo; a quick and simple technique, the E-Test®(AB Biodisk) allows a laboratory indirect estimate of the CM (figure 4).

Figure 4 : the Etest®

B- At the very methods :

1- Search beta-lactamases :

Inactivating the enzyme beta-lactam antibiotics can be detected by a chromogenic method :

The principle is based on the & rsquo; use & rsquo; cephalosporin changing color after hydrolysis. The most used is the molecule that nitrocefin, after action & rsquo; beta- lactamase, turns from yellow to red. Discs impregnated paper nitrocefin are marketed : Test Céfinase .

2- Search the méticilino resistance in staphylococci :

In standard conditions of & rsquo; susceptibility, only a fraction of the population expresses the resistance to penicillins M. only more drastic conditions (incubation at 30 ° C ; middle hyper salty MH) allow better expression of resistance. Depositing a disc d & rsquo; oxacillin to the medium surface. The reading is done 24 then 48H. A heterogeneous resistance manifested by the presence of variable size colonies around the disc. The strong presence of MRSA in the hospital environment makes their essential research.

3- Search bêta-lactamases à spectre étendu (Explosions) :

The technique is also used for dissemination within the research spectrum beta-lactamase to Extended (Explosions).

For that one has on the surface of & rsquo; agar disk ceftazidime (and / or cefotaxime, and / or cefepime, and / or d & rsquo; aztreonam) and d & rsquo; amoxicillin-clavulanic acid (for Enterobacteriaceae) or ticarcillin clavulanic acid(pourle Pseudomonas aeruginosa)

ESBLs are beta-lactamases and are thus inhibited by & rsquo; clavulanic acid. then observed a synergy & rsquo; action between the two antibiotics, called “bouchon de champagne

This technique allows to differentiate hyper-producing bacterium cephalosporinase d & rsquo; ESBL producing bacteria in the family Enterobacteriaceae such.

Vl- Special case of the automated antimicrobial susceptibility testing :

L & rsquo; s Automation & rsquo; s susceptibility & rsquo; is developed to overcome the disadvantages of this manual technique, whose realization is slow. Currently, this term is used to refer to devices performing reading and & rsquo; interpretation of tests done manually. These devices operate on 2 principles :

  • They mimic conventional tests & rsquo; study of the sensitivity of bacteria to antibiotics (The most common technique : in liquid medium dilution
  • Or they compare the growth of a witness to that observed in the presence of one or more concentrations of antibiotics.

The methods of studying the growth of bacteria employ direct optical methods :

  • Image analysis collected by a camera
  • Turbidimètrie (OD of a suspension proportional to the mass of suspended particles) ;
  • nephelometry (measuring the diffracted light). Generally, nephelometry has higher sensitivity but a narrower linearity range that turbidimetry.

VII- Synergy Events :

The use of a combination of antibiotics is justified in four cases :

-► Expanding the spectrum of activity in the case of multiple germ infections ;

-► Treat emergency undiagnosed serious infection ;

-► Prevent selection of resistant mutants during long-term treatment ;

-► Obtaining a synergistic effect.

The interaction of two antibiotics can produce four main effects :

-► Indifference : the activity of an antibiotic has no influence on the activity of the other

—► Addition : the effect of the combination is equal to the sum of the effects produced by each of antibiotics taken separately

-► Synergy : the effect of the combination is greater than the sum of the effects produced by each of antibiotics taken separately : Ex beta-lactam (or vancomycin) facilitate the penetration of aminoglycosides

-► Antagonism : the effect of the combination is less than the sum of the effects produced by each of antibiotics taken separately : Ex bacteriostatic (tetracyclines, macrolides or phenicols) decreases the bactericidal activity of beta-lactam

VIII- Conclusion :

Antimicrobial susceptibility aims to predict the sensitivity of an organism to one or more antibiotics in order to best adapt the antibiotic in an infectious context. This study takes place in vitro and should consider other features of antibiotics, Pharmacokinetic e.g., in order to have the maximum chance of recovery for the patient.

DST preparation techniques have evolved since twenty years with advances in automation. This helped to reduce waiting a view to optimizing the quality of care time, but also to better standardize susceptibility. However, old techniques have not disappeared, because the automated techniques are not perfect : anaerobic bacteria and slow growing grow poorly under techniques in liquid media. This tends to prove that a bacteriology laboratory should have two techniques to perform DST : an automatic and a manual, in order to obtain maximum complementarity between these techniques.

Cours du Dr RAMDANI Hakim – Faculty of Constantine