Isolated from saliva in 1880 by Pastor S. pneumoniae remains, despite its sensitivity to antibiotics, the first place among the causes of infectious disease death in developed countries. The discovery in 1910 of the various serotypes of S. pneumoniae had allowed the use of specific antisera which were the first effective treatment of pneumococcal pneumonia. The study of the physiology of this bacterium has led to breakthroughs that paved the way for molecular biology. In 1928, Griffith showed that strain R (rough) Non-encapsulated and non-pathogenic to mice S. pneumoniae could be transformed into a strain S (smooth), and encapsulated pathogen. In 1944, Avery, MacLeod and McCarthy laid the foundation of bacterial genetics by showing that DNA is the transforming factor among pneumococci.
I – HABITAT:
Pneumococcal frequently colonizes the human respiratory tract as there would be up to 70% of healthy pharyngeal carriers; you can sometimes find him in the genital mucosa.
This is a germ transmitted by air: the transmission is almost always direct via droplets Pflügge. The germ, deemed fragile, little survives in the environment.
It is an essentially human germ, it is rarely isolated from animals.
II – PATHOPHYSIOLOGY:
The discoveries of recent years illuminate some facts observed in infectious diseases, but does not solve everything.For a long time it was thought that the capsule was responsible for pathogenicity (Avery: Experimental pathogenicity in mice). S capsulated strain killed the mice, the non-encapsulated strain R was harmless. This is not entirely correct: there may be two encapsulated strains of serotype 3 which have the same chemical composition of the polysaccharide, the virulent, the other non-virulent for mice; the capsule is thus not the only virulence carrier.
The capsule protects S. pneumoniae phagocytosis.
The course of the disease is directly related to capsular antigens circulating rate and the absence of antibodies; we know that in pneumococcal disease alternative pathway of complement is activated and the teichoic acids of the wall would cause a cleavage of C3 in C3a and C3b units; would result in a decrease in the rate of C3 and factor B. One consequence would be disseminated vascular coagulation.
An aggregation of neutrophils can be observed, it is related to an alteration of the membranes thereof probably under the influence of pneumolysin. This alteration results in polymorphonuclear leukostasis in the pulmonary capillaries responsible hypoxia and cardiopulmonary decompensation.
Leucopenia and cytopenia spine have a poor prognosis in pneumococcal infections.
III – PATHOGENICITY:
A – Experimental:
The mouse is the animal of choice, intraperitoneal inoculation of encapsulated pneumococci results in death of the mouse. At necropsy we observed capsules germs in the blood and the organ indentations (liver …). This inoculation was used to isolate nuclei from pathological material and to identify the isolated strains.
Less and less used this mouse inoculation. This pathogenicity for mice is not constant, some serotypes (such as 14) are not pathogens, as well as strains R. Phase
B – Natural:
Pneumococcal infections can reach subjects previously healthy; but they are more frequent and severe in patients with certain immunological grounds (agammaglobulinemia, splenectomy, immunosuppressive treatment), but also in patients with impaired immunity, such as sickle cell disease, elderly …
Mortality increases with age, delay in hospitalization, the existence of associated defects (diabetes, anemia, cirrhosis, nephritis, cancer …) …
The most common sites are ENT, especially in children with type of ear infections or sinusitis; They can act as gateways to more serious infections.
1. acute lobar pneumonia:
She is very feverish, comes with a chest next point with phlegm
rusts and pulmonary signs of condensation; next to this table the classic
pneumococcal involved in post-viral pulmonary superinfection (flu
example). In pneumonia average mortality was 20%; this mortality
is higher when blood cultures are positive.
2. pneumococcal meningitis:
They occur at any age but especially in infants and the aged. They are
primary or secondary to an ENT home or head trauma; they are characterized (outside the classic signs of purulent meningitis) by a start
brutal withering, severe autonomic disorders, meningeal irritation
franc. The prognosis is poor, mortality is around 30%.
They should not be ignored, even if they are rare
– Skin gangrene
– Endocarditis, arthritis
– Genital infections …
IV – BACTERIOLOGICAL CHARACTERS:
A – Body type:
On microscopic examination, pneumococcus has an aspect in diplococci, candle flame in 8GB and short chains.
The diplococci and capped chains are Gram-positive.
However, be aware that the appearance is not always so evocative. For example, if the environment is magnesium deficiency can be observed relatively long chains. The same phenomenon occurs in the presence of antibodies against the capsular serotype. In some cases, if the patient is under treatment, one can see pneumococcal take pseudobacillaires forms.
In some pathological fibrin products and in ancient cultures, pneumococcus is poorly attached and can appear Gram Gram negative. Particularly beautiful after inoculation of mice, the capsule is usually visible in pathological products, but sometimes more discreet (Fig. 1, 2). The capsule is more visible on a preparation with the ink.
The synthesis of the capsule varies with the stage of the germ of the growth curve. The synthesis is optimal capsular end of the exponential phase and the plateau phase. When pneumococci multiply intensely, there are evil capsules;capsulaircs these polysaccharides are released into the environment, they are also released in pathological products, hence the term exoantigen soluble sometimes used to designate them.
B – Cropping characters:
The temperature range for the cultivation is 25 to 42 ° C. Routinely, the seed is grown between 35 and 37 ° C.Cultures are possible for pH situated between 6.5 and 8.3, the optimum pH being 7.8. Pneumococci in culture are subject to spontaneous autolysis; so one should try to limit this autolysis.
Employees will be rich environments, eg agar + 5% sheep blood.
On this medium, the germ develops alpha hemolysis, like its close relatives, verdissants streptococci. Some strains require CO2 in primary culture.
The strict anaerobic conditions is even better for their development and may be considered as blood agar placed anaerobically is a selective medium that promotes pneumococcus.
A macroscopic examination, colonies are in the form of small transparent colonies, round, 0.5 to 1.5 mm in diameter.
A umbilication the center of the colony is a beginning of autolysis.
Serotype III presents mucous colonies with a diameter of 3 mm, similar to Klebsiella. This mucosal appearance is due to the exuberance of the capsules.
In conditions of strict anaerobiosis, colonies are curved and of size 2 to 3 times greater than those observed in aerobic and hemolysis does not appear. By cons, if we abandon the box 30 minutes in a normal atmosphere, alpha hemolysis appear. Anaerobically in the presence of antibiotics changing the wall (penicillin, vancomycin), it is a beta hemolysis (see photo).
C – biochemical characters:
Pneumococcal has neither catalase or peroxidase, which induces the accumulation of hydrogen peroxide in part responsible for its autolysis. Other characters are:
. toumesolé milk: acidified and coagulated
. Fermentation of sugars: Acidification of glucose, lactose, raffinose, sucrose …
Two characters are more interesting:
These characters are hardly searched for the identification of the organism. Inulin, by cons, was used to differentiate pneumococcal other streptococci, but a number of streptococci verdissants can ferment inulin: Streptococcus salivarius, Streptococcus sanguis, Streptococcus uberis.
Pneumococcal formal identification is based on three criteria:
– Sensitivity to optochin and if in doubt:
– Lysis by bile,
– The detection of a capsule.
1. Sensitivity to Optochin (ethylhydrocupreine derived to quinine):
5 mm diameter discs are loaded with 5 mcg Optochin, dose calculated to induce, in a culture pneumococcus on blood agar, an inhibition zone with a diameter between 12 and 35 mm. Can be read on the manufacturers’ instructions as streptococci, by cons, are resistant to Optochin and multiply into contact with the disk. This distinction is not always so obvious. In fact, 0.5 to 5% of pneumococci are resistant and some streptococci Optochin verdissants are inhibited by Optochin. It is therefore necessary to be qualified and to take account of the diameter of the inhibition zone: most of pneumococci in S phase (smooth) or R (rough) have an area greater than 15-20 mm diameter. A diameter less than 15 mm, it is necessary to perform further testing.
2. lysis by bile or phenomenon Neufeld:
It makes a broth culture and centrifuged. The seeds were resuspended in a buffer at pH 7 and added some Na deoxycholate drop solution 2 to 10%; in few minutes, the tube clears. Bile salts activate the autolysin pneumococci.
3. Identification of the capsule:
It must have a polyvalent pneumococcal serum directed against the capsular all kinds. The reaction can be performed either from crops or from pathological products.
The capsular antigen may be revealed by “swelling” capsular if cocci are visible, either by immunological technique such as against-immunoelectrophoresis (CFFI) or technical agglutination.
D – The pneumococcal antigens:
1. Capsular antigens:
They can be identified by swelling capsular against immunoelectrophoresis or agglutination. The chemical composition of some serotypes is well known.
In 1913, it was recognized four serotypes. In 1939, we discovered 32. Can research on the development of a vaccine have revived interest in serotypes and two classifications have been proposed, one American, the other Danish. This is the Danish classification Lund prevailed; it covered 83 capsular types or different groups. It includes 27 types of antigens and 19 groups with a total of 56 type antigens.
The Statens Serum Institute in Copenhagen produced a omnisérum and 9 sera pools of A 1 and 46 monovalent sera. Determining the type or group full identification of the strain.
Pneumococci possess antigens other than the capsular polysaccharide antigens.
2. Somatic antigens:
Substance C, species specific, which is a polysaccharide consisting of teichoic acid can sometimes contaminate the capsular polysaccharide and may be responsible for cross-reactions. Its chemical composition is similar to polysaccharide C streptococci but it is different from the antigenic point of view.
The antigen R protein in nature, is often unapparent as masked by the capsular antigen.
The antigen M is a specific proteinaceous antigen pretty close type of antigen M of group A streptococci
E – Toxins:
Alongside these capsular and somatic antigens, pneumococcus develops toxins most of which are also antigenic: pneumolysin neuraminidase hyaluronidase, producer principle purpura.
1. The pneumolysin:
Responsible for alpha hemolysis is oxygen sensitive toxin, activated thiol groups, sensitive to cholesterol, cytolytic, just as streptolysin 0. It seems closely related to the bacterial body and is located intra- cytoplasmic.
Pneumolysin was purified, it has a MW of 35 kDa; otherwise its gene was cloned and sequenced.
It lysis of rabbit erythrocytes, guinea pig and human red blood cells; it also lyse white cells. Its effect as Leukocidin has long been known: the most severe purulent meningitis with low cell count. It also destroys the wafers and its toxic effect is exerted on other cells, especially those of the eye.
It has a single antigenic type and is converted into toxoid, resulting in possible applications.
2. The neuraminidase:
Many isolation strains producing this toxin which targets the sialic acids.
Purified and injected intraperitoneally to mice, it causes liver and kidney damage, and intracerebral route, causes neurological symptoms.
It can play a role in pathogenic point of view, but probably less than that played by the two previous toxins.
4. The principle of producer purpura (PPP):
It is known since 1926, but in 1981, it was shown that an enzyme intervene, either in the background or in the release of this principle: it is the N-acetylmuramyl-1-alanine amidase. This substance is not antigenic, but could reproduce or purpura, or internal bleeding.
V – BACTERIOLOGICAL DIAGNOSIS OF INFECTIONS PNEUMOCOCCAL:
A – Direct diagnosis:
The isolation of the germ is usually easier in the absence of prior treatment.
1. The levies:
The various samples to be taken for a diagnosis of meningitis, septicemia or pneumonia, pneumococcal shown in Table I.
a / tracheobronchial secretions:
Sputum are frequently contaminated with pneumococcal throat. The trans-tracheal aspirates and protected aspirations have significantly improved the quality of the sample gave the bacteriologist.
Direct examination is essential. It is indeed necessary, note the abundance of the germ and cytology. Quantitative bacteriology was proposed to do to differentiate pneumococcal the pharyngeal cavity and those of the respiratory tree, but the interpretation is not always easy.
In addition to these samples, perform blood cultures in acute pneumonia.
b / Blood cultures:
Unfortunately, they are too often neglected in the lung, so they allow, in this context, to wear a formal etiologic diagnosis (when they are positive).
Bacteremia during pneumonia is sometimes so massive that the examination of blood smears may reveal the presence of pneumococci.
Blood cultures grow rapidly; in 85% of cases they are positive on the second day, it is recommended to quickly dub the bottles to prevent autolysis germs. When suspected pneumococcal broth can be centrifuged (10 min at 2000 rpm) and perform a search for soluble antigens in the supernatant (Slidex-Pneumo-Kit).
c / liquid puncture:
– CSF: transportation cerebrospinal fluid should be done quickly and warm. In pneumococcal meningitis there is often a significant leucocytorachie and direct examination is often positive, as the number of germs will. from 3105 to 5107 CFU / ml according to the authors and countries. High microbial density combined with low cell count have a poor prognosis.
– Pleural Fluids: direct examination is sometimes evocative.
– Peritoneal fluids, joint fluid may allow the isolation of this organism.
dl Pus and various levies
Pneumococcus is often the cause of ear infections, sinusitis, it can be found from various pus and even genital swabs (urethral, vaginal …).
It can easily be obtained either by seeding liquids (broth) or solid media (blood agar) made selective by the addition of gentamicin and nalidixic acid. These environments are placed in an atmosphere of CO2 or anaerobically; some strains are very strict anaerobic isolation. The primocultures are sometimes slow and can apply 48 hours at 37 ° C.
The identification of colonies (mostly flat or umbilicated, rarely mucous membranes – serotype 3 – typically alpha-hemolytic but give beta hemolysis anaerobically in the presence of vancomycin), will be through to the test and optochin to lysis by the bile, as well as the characterization of capsular antigens (ICE or agglutination).
3. Research soluble antigens:
It can be done on pathological products (CSF, serum, urine, pleural fluids, pus …) or directly (ICE) or after prior treatment for agglutination reactions: 3 min heating at 100 ° C for CSF and urine, or decomplementation (30 min at 56 ° C) for the serum; agglutination it only considers net agglutination because the particles are highly charged (83 serotypes) have a slight tendency to self-agglutination. A diagnostic kit for research pneumococcal antigens by ELISA technique was recently introduced. Note that:
– Certain serotypes not electrically charged can not be detected by CIE.
– There are antigenic communities between S. C. pneumoniae and group A streptococci
– Can be directly serotype strains on pathological study produced by the capsular swelling, CIE or agglutination, using successively pools and monovalent sera.
This serotyping performed on strain or pathological product has a clear epidemiological interest.
B – Serology:
It could be a means of diagnosis of pneumococcal infections, but retrospective. She knows a renewed interest because it shows how a subject responds to a pneumococcal infection or how it will respond to pneumococcal vaccination. In practice, it is restricted to research.
It is to measure:
– The anti-enzyme antibody: anti-pneumolysines, anti-hyaluronidase,
– Anti-capsular antibody: but the dosage of anti-polysaccharide antibody is difficult (83 serotypes).
For the determination of these antibodies, there are ancient techniques:
– Agglutination reactions,
– Swelling reactions of the capsule,
– Precipitation reactions electro-immunodiffusion, which are replaced by two techniques that have the same performance:
– The technical radioimmunoassay (RIA) was used as the reference method for the duration of the development of the vaccine,
– ELISA has been the subject of numerous studies.
In practice we use two different approaches: determination of antipolysaccharidiques antibodies (difficult because of the multitude of serotypes) and assay of antihémolysine antibodies (pneumolysin) more promising, this assay could be of service in a perspective of diagnotic.
For these serological reactions, it is essential to have an early and late sera sera and check the rise of antibodies.
VI – PNEUMOCOCCAL ANTIBIOTIC SENSITIVITY:
The study of the sensitivity of pneumococci is not always simple. The ideal would be to determine the MIC of each antibiotic for each strain of pneumococcus, which is virtually impossible in routine. Antimicrobial susceptibility must be made, subject to respect some specific rules.
A – the susceptibility to pneumococcal technique:
– A single colony is grown first in broth enriched ascites serum for 18 hours,
– Is taken 4-8 drops of the broth which is placed in 10 ml of distilled water,
– Is inoculated by flooding a Mueller-Hinton medium supplemented with 5% horse blood and sheep
– Depositing the antibiotic disks and incubated for 18 h in an atmosphere of CO2
– One then proceeds to the reading of the diameters of zones of inhibition.
B – Results:
The main problems penicillin G: pneumococcal often lies to this antibiotic in areas of intermediate susceptibility, whereas dilution, the strain is sensitive. We can get around that. Indeed, we must know that the MIC of oxacillin pneumococcal vis-à-vis are 30 times higher
the MIC of penicillin G. In other words, if the diameter of penicillin G is low or intermediate area, it is best to measure the diameter around oxacillin (charge 5 pg) before responding to penicillin G ( Table II). This must be done systematically and must respond when a strain is resistant to oxacillin, all beta-lactam antibiotics are resistant.
As with all streptococci aminoglycosides are ineffective ;, Cephalosporins are not more active than penicillin G; the third generation cephalosporins (cefotaxime, ceftriaxone), however, have IJC quite low (0.001 to 0.06 mg / 1).
The first case of tetracycline resistance was reported in 1962. In 1967, resistance to erythromycin and lincomycin appear and in the same year, it was reported the isolation of a strain resistant to penicillin G. In 1970 publications describe strains resistant to chloramphenicol. In 1977, in Johannesburg, resistant strains of P lactams, erythromycin, clindamycin, tetracycline, chloramphenicol, and trimethoprim-sulfamethoxazole association were isolated.
In France, vis-à-vis synergistines resistance is increasing, it is stable for chloramphenicol (5-6%) to tetracycline since 1986 (about 20%) and cotrimoxazole (13-16%); but resistance is steadily increasing for erythromycin from 18.9% in 1984 to 23.6% in 1988.
Globally, the distribution of strains resistant to penicillin is very irregular (Figure 1 according Klugman) in France that resistance to penicillin G is increasing, all strains abnormal sensitivity (MIC = 0.1 mg / 1) approach 5% (3% for invasive strains, 6% for non-invasive strains) these abnormal strains are divided between mean sensitivity of strains (4.5%) and actually resistant strains (0.5%).
The mechanisms of resistance to antibiotics pneumococcal beginning to be better understood.
Penicillin: non transferable resistance not linked to the production of beta-lactamase is chromosomal origin and due to changes of the PLP.
Macrolides: cross-lincosamides-streptogramins macrolide resistance B (mlsg resistant phenotype) is mainly due to methylation of 23S rRNA which decreases the affinity of the ribosome for MLS.
It has been shown for some strains that chromosome carried a “conjugative transposon” encoding an enzyme APH (3 ‘) responsible for high-level resistance to kanamycin resistance associated to MLSb and tetracyclines, this element is transmitted how stable descent and probably plays a role in the spread of resistance.
Chloramphenicol: this antibiotic is inactivated by an inducible chloramphenicol acyltransferase not linked to a plasmid decisive.
VII – PNEUMOCOCCAL VACCINES:
A – Composition:
To deal with antibiotic resistance and to prevent mortality remains high even with the help of an antibiotic treatment, it has developed a pneumococcal vaccine. It was not possible to incorporate the 83 serotypes in the vaccine. It was therefore decided serotypes after their greatest frequency. Current vaccines have 23 valences that correspond to the following types of Streptococcus pneumoniae: 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A , 19F, 20, 22F, 23F and 33F. In this 23-valent vaccine, there are 25 u, g of each polysaccharide. He is protective vis-à-vis 90% of the strains isolated in France and Europe.
It was shown that the purified polysaccharides of the pneumococcal induce the formation of antibodies. These antibodies bind to the capsule, they attract phagocytic cells and promotes bacterial ingestion, that is to say opsonization.
Manufacturers had assayed the antibody before and after vaccination. It was necessary, for each valency, verify that there was a rise in serum antibody between an early and a late serum. It can be considered that vaccine is acceptable if there is a rise of antibodies to at least four times vis-à-vis the various antigens contained in the vaccine to 80% of subjects.
B – immunological efficacy of the vaccine:
In recent studies with commercial vaccines, it was demonstrated that 90% of subjects had a 4-fold increase in the rate of their antibodies.
The vaccine is administered at one time. It would be protective for three years, but it is not effective in children under 2-3 years. There is no cross-immunity between the 14 serotypes. It may be noted that serotype 6 is very immunogenic. It is advisable not revaccinated before 5 years, early revaccination may result in adverse effects.
Failures are observed:
– In young children, in the elderly,
– In Hodgkin’s disease,
– After splenectomy, so it is preferable to vaccinate candidates for splenectomy before it,
– In the nephrotic syndrome where the results are often misleading,
– In hematological, the answer is not always good; by cons, it is correct in sickle cell disease.