Antibiogramme
A antibiogramme is a technique of laboratory aiming at testing the sensitivity of a bacterial strain with respect to one or several Antibiotique S supposed or known.
The principle consists in placing the culture of bacteria in the presence of or antibiotics and observing the consequences on the development and the survival of this one. One can for example place several pastilles soaked with antibiotics on a bacterial strain deposited in a Boîte of $petri. There exist three types of interpretation according to the diameter of the circle which surrounds the disc of antibiotic: stock or sensitive, intermediate or resistant bacterium.
Realization of a antibiogramme
Material
- a gélose Mueller-Hinton out of box of $petri
- discs of antibiotic, or a distributer allowing the demounting standardized of the discs on the gélose.
- a pure stock of the bacterium studied
- a rake or a flue brush
- a pipette of 1 ml
- tube with hémolyse
- pipette Pasteur
- standard of Mac Farland n°0.5 (1.5*10 ⁸ UFC/mL)
- sterile physiological Water
Stages
Realization of a suspension
two possible solutions:-
is you have an old culture medium of 24:00 (stationary phase) you can use it in the following way:
- for thepositive one, to carry out a dilution to the 1/500;
- for thenegative one, to carry out a dilution to the 1/5000.
- is you have pure colonies on a culture medium, in this case:
- to fruitlessly put physiological water in a tube at hémolyse;
- to take the pure colonies and to put them in suspension until obtaining same opacity as the standard Mac Farland 0.5;
- if the suspension is too turbid, to adjust opacity by adding physiological water.
Preparation of the gélose
- to take the gélose of Mueller-Hinton, to check the absence of water on the surface; if there is, to let dry;
- to annotate where will be positioned the antibiotic discs on the bottom of the box (It should be moved away from 1 cm of the minimum edge);
- (council: to divide the box as much once (maximum 5 for a box of 10 cm, or 12 for a box of 15 cm) which you have of antibiotics, or to use an owner);
- to sow the gélose by 1 ml of suspension;
- to spread out volume with the rake of the center towards the edges;
- or to soak the flue brush in the suspension, to remove the excess of inoculum by pressure on the edges of the tube, to clean the gélose regularly while turning the plate of 60° until sowing of the totality of surface;
- to let dry from 3 to 5 minutes;
- to deposit the antibiotic discs;
- to incubate 16 with 18:00, 35°C (to the 24:00 maximum).
Reading of the results
- To measure the diameters of the aureoles (zones of inhibition of growth of the microbial stock).
For each microbial stock, the sensitivity or resistance to an antibiotic is different. It calls upon the concepts of concentration criticizes lower (c) and of concentration criticizes higher (C).
-
C: minimal antibiotic amount that a patient can receive without dangers and which makes effect on the bacterial strain.
- C: maximum antibiotic amount that a patient can receive without dangers and which makes effect on the bacterial strain.
Pharmacokinetic: in somebody correctly treated by an antibiotic, the antibiotic concentration in the organization is supposed to oscillate between the concentration criticizes lower and higher.
These data are available on abacuses.
For each couple bacterium-antibiotic, one determines an inhibiting minimum concentration (or inhibiting minimal concentration, or CMI). The CMI are the smallest antibiotic concentration which inhibits any visible growth. By comparing the CMI with the critical concentrations, one determines the sensitivity or the resistance of the bacterium to antibiotic.
- the bacterium is sensitive to antibiotic when the CMI are lower than the concentration criticizes lower. Concretely, this means that it is enough to a weak antibiotic concentration to kill the bacteria and that this amount necessary is even weaker than weakest of the amounts which one can manage at the man. Thus in light, if one treats somebody with antibiotic, the concentration of this one in the organization will be always sufficient to kill the bacteria.
- the bacterium is resistant to antibiotic when the CMI are higher than the concentration criticizes higher. Concretely, the amount necessary to kill the bacteria is well too high to be supported at the man without important side effects. This antibiotic cannot be used to treat an infection.
- the bacterium is intermediate with antibiotic when the CMI lie between the two critical concentrations. In practice, that corresponds to a situation where the concentration is sometimes sufficient to kill the bacteria, sometimes insufficient. It should be considered that the bacterium in vivo and should not be used this antibiotic will be resistant.
Reading criticizes antibiogramme
A antibiogramme must be interpreted insofar as the results in vitro are not always reproducible in vivo . For example, if a Staphylococcus aureus (gilded Staphilococcus) is resistant to the oxacilline (in the past méticilline), it should be considered resistant to all the beta-lactam antibiotics (all penicillins and all the cephalosporines become unusable). One calls such a bacterium a SARM ( Staphylococcus aureus resistant to the méticilline, English MRSA).
The use of the antibiogramme to the daily newspaper
The antibiogramme is a tool validated by a medical biologist who allows a doctor to choose good antibiotic, or the antibiotic association making it possible to treat a patient effectively. However, most of the time, it is not necessary because much of infections are effectively treated in a probabilistic way. One makes an assumption on antibiotic and the bacterium to be treated according to the place of the infection (amygdala, abdomen, lung…) because they are often the same species that one finds at the same place. A antibiogramme is made in the case of serious infections (shock septic, infections nosocomiales…) where when the antibiotics chosen as a probabilist do not function. This attitude leads to the repeated use of same antibiotics, and contributes to the emergence of resistant stocks.
Taking away with the antibiogramme
The bacteria are collected:- in blood. They are then put in culture (hémoculture),
- in the urines,
- in the saddles, the excrements,
- in spittles, bronchoalveolar washings (one washes with water the bronchi during a fibroscopy and one analyzes this liquid washing),
- in the céphalo-rachidian liquid, in which bathes the brain, the cerebral trunk, the cerebellum and the spinal-cord,
- …
The realization practices antibiogramme
- the antibiogramme is often automated. The bacteria are deposited in suspension (in a liquid) in a container, then the machine aspires this liquid and deposits it on a chart (format credit card) in which there are small zones corresponding to each antibiotic. The machine reads then this chart and gives the results automatically (sensitive, intermediate, resistant).
- Sometimes, one can remake a antibiogramme for certain antibiotics chosen by the biologist. Either one carries out the procedure described into 1. Either one uses other more expensive, but simpler techniques like the E-test. A box of $petri is sown, then one deposits a band E-test which includes/understands an increasing antibiotic gradient from one end to another (not of antibiotic on the left, maximum concentration on the right) and these antibiotic concentrations are written directly on this tape. One puts in incubation the box and one reads the CMI with the intersection of the disc of inhibition with the band.
Interpretation of the antibiogramme
- the antibiogramme is not returned such as it is. An interpretation of the raw data is necessary.
- resistances of the bacteria are not always expressed. It is thus important to know natural resistances.
- Certaines resistances is heterogeneous and will touch only part of the stocks. In this case, it is sometimes necessary to work under conditions which support the expression of resistance.
- the study of the mechanisms of resistance makes it possible to establish profiles of resistance.
- the antibiogramme is also a good tool of orientation for certain stocks difficult to identify.
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