Antibiotics and antibiotic susceptibility


I- Historical :

The first antibiotic, identified from the late nineteenthe century by Ernest Duchesne, was penicillin. Its properties were rediscovered by chance in 1928 Sir Alexander Fleming s & rsquo; saw that some of his bacterial cultures in forgotten Petri dishes were contaminated by the experiences of his neighbor bench on a mushroom : le marked Pénicillium. But the importance of this discovery, its implications and medical uses were not understood and developed until its rediscovery, between the two great wars.

The first antibiotic (of synthesis) has opened a new path in the fight against many diseases that were previously considered incurable. Antibiotics have increased the & rsquo; life expectancy of those who have access to d & rsquo; about 15 years. comparatively, a medicament that would cure 100% cancers would increase the & rsquo; life expectancy of 5 years.

II- Definition :

(Greek anti : " against ", and bios : " life ") is a substance that has a specific action of blocking or even killing bacteria. For other microorganisms, we use the term d & rsquo; 'antifungal' s & rsquo; s it & rsquo; is to fight against fungi, or "viral" if it is to fight against viruses.

This substance can have a direct toxic effect, c & rsquo; is to say bactericidal ; its effectiveness can be limited to prevent the growth of microorganisms (bacteriostatic).

– WAKSMAN (1943) :toutes les substances chimiques produites par des micro-organismes capables d’inhiber le développement et de détruire les bactéries et d’autres micro-organismes

– TURPIN AND HAIRY (1957) : ” Any chemical compound, produced by a living organism or produced synthetically, high chemotherapeutic coefficient whose therapeutic activity is manifested at very low doses of & rsquo; Specifically, by inhibiting some vital processes, against viruses, microorganisms or even some multicellular beings ''.

III- Classification :

It is necessary to know the family's general characteristics and distinctive advantages of the different members & rsquo; family.

  • The 11 families of antibiotics are :

•Bêtalactamines : penicillins / • Aminosides

  • Phenicols • Tetracycline
  • Macrolides & Related
  • Polypeptides
  • Sulfamides
  • Quinolones
  • Nitro-imidazoles
  • Derivatives nitrofurans
  • kernel derivatives Benzyl – Pyrimidine.

Antibiotics are classified into families from a number of criteria :

  • Origin : produced by a living organism or produced synthetically, At & rsquo; now, often it will molecules, usually obtained by semi-synthesis.
  • chemical nature : very variable, often a basic structure as the B-lactam (family of B-lactams) on which there semisynthetic.
  • Modes & rsquo; Action : Their mode of & rsquo; work though sometimes imperfectly known, is d & rsquo; great variability, or complex. His knowledge may help to understand the synergy and the natural resistance mechanisms and acquired.
  • Terms of & rsquo; Action : is the in vitro study of the interactions in time between varying concentrations of & rsquo; antibiotic and & rsquo; a bacterium.
  • Spectrum of activity : List of Species in which antibiotics are active (spectrum narrow or wide). The spectrum will be discussed during the & rsquo; study therapeutic classes.

Example :

IV- User & rsquo; Action :

The action of antibacterial antibiotics mechanism is not always fully understood but one distinguishes five main modes of action :

  • Action on peptidoglycan synthesis.
  • Action on the cytoplasmic membrane.
  • Action on DNA.
  • Action on protein synthesis.
  • Action by competitive inhibition.
Targets of antibiotics

  • Les β lactamines : Acting in the calf or bacterial cell wall by inhibiting the final stage beyond peptidoglycan synthesis resulting in bacterial lysis.
  • aminoglycosides : Disrupt protein synthesis at the ribosome fraction 3os resulting bacterial destruction. They are bactericidal.
  • the phenicols (chloramphenicol and thiamphénicolé) : Both molecules are bacteriostatic. They act at the subunit 50 S du ribosome. This results in inhibition of protein synthesis.
  • Les tetracyclines : Inhibit protein synthesis at the subunit level 30 S du ribosome.
  • the polypeptides : are molecules that n & rsquo; act only on gram-positive bacteria by inhibiting peptidoglycan synthesis therefore of bacterial growth.
  • Macrolides Related : Macrolides act by inhibiting bacterial protein synthesis. They attach to the & rsquo; unit 50 S ribosome and block and the meeting of the last stage of the synthesis. They are bacteriostatic.
  • Les Quinolones : Inhibent la synthèse del’ADN de la bactérie en se fixant sur le complexe “ADN- ADN gyrase” en empêchant la réplication et transcription de l’ADN bactérienne.
  • Sulfonamides and associations : They have a static activity b actério. They compete with the P AB (p ara-aminobenzoic acid) thus blocking the & rsquo; synthetase action.
  • Les Nitrofuranes : Act by disrupting replication of & rsquo; DNA.
  • the Nitromidazoles : They act by inhibiting nucleic acid synthesis resulting in the rapid death of the bacterium. The nitroimidazoles are bactericidal.
  • Les Rifampicines : Work by blocking transcription by inhibiting & rsquo; RNA polymerase.

NB : TB are characterized by their bactericidal power on the bacillus (B.K.). In Algeria, Rifampicin, l & rsquo; Isoniazid, l’Ethambutol, Streptomycin and Pyrazinamkle are reserved for the treatment of tuberculosis (emergence of chromosomal R mutants).

V- Antibiotic resistance mechanisms :

1 – The types of resistance :

Are known natural resistance, programmed into the bacterial genome, therefore fixed and constant at & rsquo; inside the taxon. To this, they constitute a criterion for & rsquo; identification.

Are known acquired resistance, consecutive to changes in the & rsquo; chromosomal gene or plasmid equipment. They concern only a few strains of & rsquo; the same species but can s & rsquo; expand : their frequency varies in time but also in the & rsquo; space – region, city, hospital or same service. They are an epidemiological marker.

2- resistance phenotypes :

When we study the sensitivity of & rsquo; a strain to several antibiotics, its antibiotic resistance phenotype is determined. If the strain n & rsquo; expressed as natural resistance, on dit qu’elle appartient au phénotype “wild” or sensitive. If the acquired resistance has changed its sensitivity, it expresses a phenotype of resistance that & rsquo; can be identified and which one should attempt to determine the mechanism. These phenotypes are often designated by the initials have become inactive antibiotics : and a strain resistant to kanamycin, tobramycin and gentamicin belongs to KTG phenotype.

3- The resistance levels :

D & rsquo; a bacteriological, it is said that & rsquo; a strain is resistant when & rsquo; it can grow in the presence of & rsquo; a concentration of & rsquo; antibiotic higher than the concentration which inhibits the majority of strains of the same species. We must therefore consider d & rsquo; a dose effect. There is talk of low resistance if growth is blocked by low concentrations of & rsquo; antibiotic and high resistance if high concentrations are needed.

4- Genetic support resistance :

Natural resistance is programmed into the bacterial genome. Acquired resistance to genetic changes are responsible chromosomal, secondary to a mutation on chromosome or extra-chromosomal genes by acquisition.

Thus bacteria defend themselves against the action of antibiotics by three mechanisms :

– By being impervious to penetration.

– Producing enzymes capable of inactivating the.

– By changing the structure of their target.

WE- Study of the susceptibility of bacteria (The Antibiogramme) :

L & rsquo; susceptibility testing is to determine the Minimum Inhibitory Concentration (CMI) a bacterial strain vis-à-vis the various antibiotics. By definition (O.M.S.), MIC is the lowest concentration of & rsquo; antibiotic capable of causing a complete inhibition of the growth of & rsquo; a given bacterium, appreciable to the naked eye, after a period of & rsquo; given incubation. The determination of this value is not very accurate but it is time-honored and enjoys d & rsquo; a large mass of & rsquo; information gathered about him.

a- Definition:

Is a laboratory technique to test sensitivity d & rsquo; a bacterial strain vis-à-vis one or more known or suspected antibiotics.

b- Methodology :

1- 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 the 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 antibiotic and wherein no growth is visible.
MIC determination by dilution in liquid medium

– In mid strong 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 (inoculator a multihead, apparatus Steers, used to inoculate 20 at 30 different strains per box). after incubation, MIC of each strain is determined by the & rsquo; inhibition of growth on the medium containing the lowest concentration of antibiotic.

Determination beyond MIC by agar dilution

In current practice, dilution methods are put in delicate work and / or expensive and are reserved to specialized laboratories.

2- 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. A petri dish 9 cm diameter allows deposition of ôdisques impregnated & rsquo; antibiotics. after incubation, CD s & rsquo; surround areas & rsquo; circular inhibition corresponding to an absence of culture

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- special techniques :

  • E- test (Using strips impregnated with anti-tuberculosis for a quick test in mycobacteria).
  • Antibiogramme automated or automated.

5- Definition of bacteriostasis and Bactericidal :

MIC = Minimum Inhibitory Concentration : Concentration & rsquo; antibiotics lowest inhibiting any visible croissance_bactérienne (Widely used in practice).

MBC = Minimum Bactericidal Concentration : Concentration & rsquo; Antibiotic leaving less 0,01 % survivors.

Rapport CMB/CMI :

Used to distinguish

  • bactericidal antibiotics (CMB/CMI < 2)
  • bacteriostatic antibiotics (CMB very far from the CMI)

Defines the tolerance of strain (CMB/CMI >32)

Ex. Gram-positive bacteria and ATB assets on the wall (Streptococci and b-lactams).

6- Expression of results :

The quantitative results (CMI en mg/mL) are usually performed by laboratories in terms of therapeutic possibility. this interpretation, usually required by clinicians, is, according Chabbert, neither compulsory nor always desirable. It involves comparing the MIC values ​​with the critical concentrations established for each antibiotic.

After inoculation and incubation, the disc & rsquo; amoxycillin is surrounded by & rsquo; an area & rsquo; inhibition 10 mm (measurement using calipers).

schematically, the upper critical concentration corresponds to the greatest amount of & rsquo; active antibiotic as & rsquo; can be obtained in the serum and tissues as a result of a treatment carried out at the usual dose and the lower critical concentration corresponds to the lower humoral concentration and tissue d & rsquo; active antibiotic.

7- 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. Moreover, for some couples bacterium-antibiotic, despite categorization ” sensible “, the increased risk of in vivo selection of resistant mutants justifies special comments for the clinician. The interpretive reading requires correct identification of the strain and a method & rsquo; perfectly standardized susceptibility testing. The detection of resistance phenotypes highly unlikely 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.

8- Limitations of the & rsquo; susceptibility :

The realization of & rsquo; antibiotic sensitivity is subject to compliance with technical conditions that are sometimes incompletely and inadequately respected.

Susceptibility testing must be performed on a pure and identified Culture. This last condition allows d & rsquo; adjust the density of the & rsquo; inoculum, judicious selection antibiotics to be tested and to practice interpretive.

A susceptibility testing of non-standardized manner and on a mixture of unidentified bacteria is meaningless.

Any laboratory should confirm the validity of his technique by testing, at least once a month, the reference Susceptibility and verify that the diameters of the zones & rsquo; inhibition obtained vis-a- vis various antibiotics are consistent with the values ​​published by the Committee of the & rsquo; Antibiogramme. The techniques of & rsquo; susceptibility were standardized only for cultivating bacteria quickly on usual media. When the isolated strain does not fall within this framework, l & rsquo; interpretation is sometimes difficult.

9- Example : Search heterogeneous resistant Staphylococcus :

The highlight of this particular resistance to Staphylococci which only a fraction of the population expresses the character vis-à-vis the beta lactam (SARM), poses bacteriologist at a delicate problem. The susceptibility of conventional medium not usually allow its detection.

Various technical devices make possible unmasking the phenomenon. an inoculum is used germs busier than usual, a culture medium said hypertonic consisting of a Mueller Hinton medium containing 5% Na Cl. The reading is made after incubation for 24 h at 30 ° C, confirmation is done after 48 hours, the existence of a heterogeneous population known results in bacterial regrowth made settlements of varying size around the penicillin disk M, while a single disc could, under normal conditions showed a good zone of inhibition.

VII- Conclusion :

In less than a century of antibiotic has become an effective way to fight against infections, but the abusive and uncontrolled use constitutes a danger to the population and a headache for the scientific community begins to pull the alarm to raise awareness of the risks involved and with dramatic consequences in the absence of a therapeutic substitute.

Courses of Dr. H.ALLAG – Faculty of Constantine