Actinomycetes

Actinomycetes are in the order of Actinomycetales. Some representatives of the actinomycetes, especially among aerobic, have long been rejected by all the bacteria and fungi mistaken because of their morphology, sometimes fungoides. This is a suprageneric group, bringing together diverse bacteria dispersed in systematics, where one brings Nocardia genera Actinomyces, Mycobacterium, Corynebacterium, Streptomyces actinomycetesBifidobacterium … These are important for the microbiologist, it is the agents of human or animal disease (actinomycosis, nocardiosis, tuberculosis, mycetomas …) abundant in nature (soil, water, compost). They play a key role in soil fertilization in the biodegradation of organic compounds or pollutants (pesticides) in certain plant diseases; they are finally at the origin of many antibiotics.

For the sake of simplification, and for the practice of medical bacteriology, we are considering in this chapter that genres interesting human pathology and not described in other specific chapters:

– Aerobic actinomycetes (Nocardia, Actinomadura, Streptomyces)

– Strict or facultative anaerobic actinomycetes (Actinomyces, Arachnia).

HISTORY:

Actinomycosis was first described in cattle and humans; the first culture obtained in humans is due to Israel in 1878.

In 1888 Nocard, French veterinarian, described farcy a cattle infection resembling chronic tuberculosis.

Eppinger observed in 1890, the first case of nocardiosis pseudo-tuberculosis allure associated with brain abscess.

In 1940, Erickson recognizes several species among the anaerobic microorganisms: Actinomyces israelii, A. bovis …

Waksman in 1959 suggests a classification distinguishing strictly anaerobic Actinomyces or sometimes optional,Nocardia and agents of mycetoma, aerobic.

I – CLASSIFICATION:

In Bergey’s Manual 1986 (vol. 2), the Arachnia and Actinomyces genres are classified as irregular bacilli section, non-spore forming Gram-positive, not as strict anaerobes, but as facultative anaerobes but added that most species grow best under anaerobic conditions (at least in isolation).

The 1989 edition of Bergey’s Manual (Vol. 4) is devoted entirely to actinomycetes (HA Lechevalier).

The suprageneric groups actinomycetes where we meet interesting germs directly medical bacteriology are:

Actinobacteria Nocardioform

Micrococcus Corynebacterium

Mycobacterium Rothia

Stomatococcus Nocardia

Oerskovia Streptomyces

Actinomyces Streptomyces

Arachnia

Brevibacterium

   Dermatophilus

Maduromycètes

   Actinomadura

II – HABITAT – TRANSMISSION:

Actinomycetes “anaerobic” are commensal bacteria forced natural cavities of man and the higher animals (mouth, digestive tract especially at the terminal ileum and cecum).

They are part of the flora of Veillon.

Unlike aerobic actinomycetes are not found in the soil or water. However, there is higher incidence in rural areas (vector role of cereals has been mentioned) and in the oral cavity subjects with poor oral hygiene.

Contamination is usually endogenous, but after bite cases have been reported and rare cases of suspected orogénitale transmission.

The front door is frequently oral (pyorrhea) or ENT, gynecologic sometimes. Then there is abscess formation, which tend to diffuse locoregional way (for example tubo-ovarian), but also sometimes with blood dissemination (including portal).

The “aerobic” Actinomycetes are widely distributed in soil, plants, water and salt and gifted in the atmosphere.

The nocardiosis is mostly contracted by inhalation, more rarely by digestive or skin.

In the case of mycetoma is generally accepted that the germ develops from an injury or a bite of a soiled piece of earth or dust.

III – PATHOPHYSIOLOGY:

In actinomycosis, there is the initial stage a badly limited infiltration with granulocytes and softening and fistula with abscess appearance or in typical cases pus yellow grains. Lesions are avascular evolving by contiguity, through blood or lymphatic system, with aspect sclerosis (limiting lesions but promoting anaerobic bacteria and protecting) and necrosis. The lesions may take a pseudotumoral appearance. The characteristic feature is the formation of yellow or white grains: mycelial mass corresponding to a complex calcium-rich polysaccharide-protein.

Officers nocardiosis and mycetoma have limited pathogenicity, evolution is slow and insidious, but can lead to spectacular soft tissue injuries and bone.

In nocardiosis, bacterial filaments occur in free form whereas in mycetoma found grains consist of aggregated filaments whose color can orient the histological diagnosis.

IV – PATHOGENICITY:

A – For the animal:

N. asteroids inoculated intravenously or intraperitoneally causes in guinea pigs of pluriviscérales damage and death of the animal in 2-4 weeks, with the presence of grain histological examination.

Pathogenicity is difficult to reproduce with Actinomycetes “anaerobic”; there is a natural power pathogenic to the animal, A. bovis causing head and neck lesions in cattle.

B – In humans (Table I):

1. Real actinomycosis are underestimated due to the difficulty:

to make the diagnosis, to link the isolation of the organism to the pathology of the frequent association with other anaerobic and / or aerobic.

The actinomycosis are favored by several factors:

– Local trauma,

– Changes in the gastrointestinal mucosa (bones, bones …)

– Presence of foreign bodies (eg intrauterine)

-Land immunocompromised steroids …

Indeed actinomycetes spontaneously low virulence.

These actinomycosis are polymorphic affections can evolve on an acute or chronic fashion and with fever and leukocytosis. We distinguish schematically:

– Forms of head and neck (50% of cases). They follow a home or tooth extraction. Swelling or jaw or neck parotid invading the skin and evolving acutely or chronically with exit pus yellow grains fistula or the puncture site; changes can be made to osteitis;

– Chest forms (25% of cases). The origin is usually gum or

tonsillar. Changes may take pseudotuberculeux pseudotumoral or appearance;

– The rarest abdominal locations. We find a appendiceal origin, gallbladder, perforation;

– Other forms can meet: cerebromeningeal, eye, liver simulating carcinoma, urogenital (increasingly frequent due to intrauterine devices), vertebral, peritoneal … and sometimes occurs a few months after a bite.

2. nocardiosis:

They are infrequent, but surely increasing. These cosmopolitan infections are favored by antibiotics, steroid treatment, cytotoxic agents, immunosuppression, organ transplants.

– The nocardiosis N. Asteroids is essentially a lung disease.

Spread is by blood from the primary pulmonary focus.

– We meet pleural localization, brain, spleen, kidney, heart, liver …

– Forms subcutaneous exist; they appear as polyfistulisés ulcer and abscess.

3. Mycetomas:

They are found in tropical and warm temperature. Predominance is noted among male adults.

The incubation period following the trauma can last for months or even years. The trend can last up to 20-25 years.

The appearance is that of evoking pseudotumor abscesses affecting the subcutaneous tissue. Granulomatous lesions can reach the bone by destroying it. The lesions are often located at the feet where the “Madura foot” is often used.

The etiology is suggested by the presence of grains in pus (white, yellow or red, variable in size, not always visible to the naked eye).

V – BACTERIOLOGICAL CHARACTERS:

 

A – morphological characters:

These are Gram positive bacilli from 0.2 to 1 micron wide with lengths varying from 1.5 to 50 microns may be in the form of filaments, bacilli or coccobacilli. Some species retain the Gram either uniformly or so irregular.

Staining with methylene blue can be observed more easily the morphology of the filaments, connections (includingD. congolensis) sporulation bodies. Some species (7v. Asteroids, N. brasiliensis) are acid-fast when using a variant of the Ziehi staining.

The various Actinomycetes exhibit differences in composition of their wall.

B – Cropping characters:

1. anaerobic Actinomycetes:

Are used Columbia agar enriched with 5% horse blood, optionally added nalidixic acid.

Cultures maintained anaerobically are examined after 2 and 7 days. Growth is favored by CO ^.

After one week, colonies appear opaque, white, irregular, with a central crater “molar tooth.” Agar, examination of young colonies shows branched filaments: characteristic mycelial growth; depending on the species growth occurs in anaerobic or microaerophilic, or optional in strict anaerobic conditions.

2. aerobic actinomycetes:

Agar Sabouraud glucose-represent the medium of choice (inclined tubes, blocked), but also allow blood agar culture under aerobic conditions.

Liquid media: thioglycolate, brain heart allow any enrichment; in these environments crops appear as veils, lumps.

Note that Nocardia may, in part, resist decontamination treatments of pathological products used for isolating mycobacteria; they thrive on Loewenstein. Colonies appear depending on the species in 3 to 5 days at 37 ° C under 10% CO2; these colonies are often pigmented, they are flat, sometimes raised dome in peak, they can be folded or waxy or cerebriform. The appearance is sometimes fuzzy. Colony morphology varies done with the species and age of the colonies.

The slide cultures allow the observation of mycelia, branched filaments, and reveal more complex formations: N.asteroids with sporulation Arthrospores type sporulation short chains …

C – biochemical characters:

1. anaerobic Actinomycetes:

We can use APIs-Anaer galleries, with varying results, or API-Zym gallery provided you use dense suspensions.

One can also use the conventional galleries:

– Study of hydrolyzed gelatin (Frazier), starch and esculin, nitrate reductase, indole, urease

– Study of arabinose fermentation, glucose, sucrose, maltose, mannose, mannitol, lactose, xylose.

Analysis of volatile fatty acids by gas chromatography (ac. Propionic …) broth products PGY allows you to make a differential diagnosis element with other Gram-positive bacteria flora of Veillon (Eubacterium, Propionibacterium … ).

The observation of the mycelial growth of blade is an orientation to the diagnosis should not be overlooked.

For Arachnia propionica, the detection of DAP in the wall is important.

The main differential characters are shown in Table II.

2. aerobic actinomycetes:

For the identification of the main species, the following characters are studied:

– Hydrolyzed gelatin, casein, tyrosine, xanthine, adenine and esculin, nitrate reductase, urease,

– Research acidification arabinose, xylose, rhamnose, adonitol, lactose, mannitol … The differential characters are shown in Table III.

The use of culture media in paraffin for isolating Streptomyces and Nocardia from pathological material, highlighting mycolic acids (C40-C60) of wall of Nocardia (HPLC, GC) and isomers diaminopimelic acid (DAP) in thin layer chromatography of cellulose (Nocardia : Meso-DAP, Streptomyces: LL-DAP), are the kind of diagnostic elements.

VI – BIOLOGICAL DIAGNOSIS:

A – samples:

– Pus fistula or punctures (pleural, bone …) pus diverse: ENT, tear, pelvic, abdominal …

– Biopsies lung …

– Intraoperative (pseudotumor), or post-mortem.

For Brown, 88% of diagnoses are established through surgery.

B – Review of pus:

The appearance of pus with grains, appearance and color them (when they exist) can suggest the diagnosis.

1. Pathology:

This examination can sometimes evoke or even practically confirm the diagnosis of actinomycosis, although typically only bacteriology allows confirmation of the diagnosis.

In lesions (swelling fluctuating pseudotumoraux aspects), two characteristic elements are found almost steadily:

– The actinomycosic follicle with a rich central area of ​​polynuclear altered, and necrosis, and periphery vascular tissue infiltrated with inflammatory cells,

– Actinomycosic the grain in the center of this non-specific inflammatory reaction. It is oval, rounded, sometimes lobed, cracked or broken, with a size of 10 to 30 microns. These grains are colorable PAS.

Other colors are used haematoxylin-eosin-saffron, Gram, silver staining (Gomori Grocott).

2. Bacteriology:

– Direct examination is necessary, it allows:

– To evoke a actinomycetes (filamentous aspect, branched, with bulges …)

– Clarify the appearance mono or polymicrobial pus.

We use Gram staining, Giemsa ZiehI-Neelsen, PAS.

– Culture includes seeding in parallel:

– Liquid enrichment media (thioglycollate medium, TGY media or PGY added serum, brain heart

– Solid media box (Columbia agar horse blood, blood agar nalidixic acid agar VCF …) placed for some aerobically for other anaerobic. Parallel were seeded tubed media: Sabouraud glucose and mid Loewenstein. Incubation at 37 ° C, monitoring for 3 weeks.

Is carried out on a culture plate to observe the 40 x 10 on the edge of young colonies branched filamentous morphology.

Once the pure strain obtained, it verifies morphology, aerobic or anaerobic characters, then we proceed to an identification through biochemical characters or antigen in the case of Actinomyces israelii.

There is no serodiagnosis. The complement fixing antibodies and precipitants are not specific.

VII – TREATMENT:

Surgery often precedes medical treatment because the diagnosis is sometimes posed as the only pathological postoperative examination.

The mainstay of treatment is antibiotics alone or associated with surgery.

A – Anaerobic:

Penicillin G is the most widely used antibiotic, optionally in combination with probenecid. Note that of A. Israeliiresistant to penicillin G strains were isolated during prolonged treatment.

In case of allergy, is used to tetracyclines, lincomycin, clindamycin, or chloramphenicol.

Actinomyces are resistant to metronidazole, the trimethoprim-sulfamethoxazole combination has an irregular action aminoglycosides are inactive.

Treatment must be intensive and prolonged several weeks.

B – Aerobic:

1. Nocardia, Actinomadura and Streptomyces:

Nocardia and Streptomyces are multiresistant to antibiotics bacteria (the majority of B-lactam antibiotics and cephalosporins are ineffective). Moreover, the retarded growth of these microorganisms often makes it difficult to interpret antibiogram diffusion of antibiotics into agar disks. Sulfadiazine and sulfonamides have long been considered the treatment of choice. Presently preferred association trimethoprim-sulfamethoxazole would give 90% treatment success. Results were also obtained with amikacin, minocycline, sulfone mother. Recent molecules cefotaxime, ceftriaxone and imipenem are active in vitro, but it lacks therapeutic data (note, in Nocardia, the presence of penicillinase inhibited by clavulanic acid).

2. Dermatophilus:

The penicillin G-streptomycin association is active, but other antibiotics act at least in vitro: chloramphenicol, tetracycline, erythromycin, but not griseofulvin.