Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria à Chapter 6: Aerobic Gram-positive filamentous bacteria
Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria
Chapter 6: Aerobic Gram-positive filamentous bacteria
Author: Dr. M.M. Henton
Licensed under a Creative Commons Attribution license.
Table of CONTENTS
Table of CONTENTS 1
INTRODUCTION 2
Table 6.1: Appearance of pathogenic actinobacteria in exudates or tissue smears/sections 3
Table 6.2: Gram-positive bacteria showing slight branching 4
Table 6.3: Gram-positive bacteria showing extensive branching (A= aerobic, F= facultative anaerobe) 7
Dermatophilus Congolensis 8
Nocardia 11
Table 6.4: Hydrolysis and selected biochemical profiles of pathogenic species of Nocardia 13
Table 6.5: Differentiation of the causative agents of canine nocardiosis and canine actinomycosis 14
Streptomyces 14
Table 6.6: Identification of Actinomyces spp. (usually glucose positive) 15
REFERENCES 16
Table 6.7: Differential properties of A. kentuckyensis, A. lexingtonensis and A. pretoriensis (agents having caused abortion in mares) compared with previously described species of the genus Amycolatopsis 17
APPENDIX 1 18
INTRODUCTION
The class Actinobacteria comprises a heterologous group of bacteria that have the ability to form Gram-positive, branching filaments of less than 1 µm in diameter. Fungi are eukaryotes and their hyphae (filaments) are greater than 1 µm in width. The main animal pathogens in the class Actinobacteria are in the genera Actinomyces, Actinobaculum Mycobacterium, Corynebacerium, Arcanobacterium, Rhodococcus, Nocardia and Dermatophilus. Nocardia is closely related to the genera Corynebacterium, Mycobacterium and Rhodococcus, all 4 of which belong to the family Corynebacteriaceae. Some members of the Actinobacteria are not invasive but inhalation of their spores can cause allergic pulmonary disease in man and horses and possibly other domestic animals fed or exposed to mouldy hay in the same way as some fungi.
The general characteristics of the genera Actinomyces, Nocardia, Rhodococcus and Dermatophilus as well as genera that can be easily confused with them are presented in Tables 6.1, 6.2 and 6.3
Morphological identification
Distinguishing pathogenic Nocardia and Actinomyces from common contaminants such as Streptomyces, Agromyces and Oerskovia is difficult, particularly in a veterinary laboratory, as many similar contaminants occur in soil or compost and from animal skin. Identifying unique members such as Dermatophilus is relatively easy, but the others require specialized techniques such as high-pressure liquid chromatography analysis to identify cell wall amino acids, sugars and mycolic acids. These techniques are unavailable to the normal clinical laboratory.
The best approach is to examine the morphology very carefully on a variety of growth media and at different stages of growth, in the same way that fungi are identified. Both the colonies and stained smears should be examined. Liquid cultures may also be examined, but the entire tangled mass of growth may only be present as a pellicle, deposit or flakes, too dense to visualize adequately.
An ordinary light microscope is required, preferably with a long working distance lens, to prevent fogging. Colonies may be examined directly, at all stages of growth, by placing the closed Petri dish containing a clear medium on the stage. If a pathogen is suspected, it is better to examine the dish upside down, through the medium. A rich medium, such as blood or serum agar, is not recommended as this allows this group of bacteria to grow rapidly and atypical growth may occur. Spores may be absent, the culture may lack an aerial mycelium or rapid fragmentation may occur. Rapid growth often results in a leathery colony, difficult to examine. A poor medium, such as tap water agar, oatmeal agar or inorganic salts agar is required for the more robust species, but nutrient agar may be sufficiently poor for Actinomyces spp. The bacteria grow slowly on these (7 - 30 days) and therefore the agars should be thickly poured to avoid desiccation. Growth is more typical on poor media, and it is easier to prepare smears, as the growth is more butyrous. The disadvantage is that the slower rate of growth takes far more time.
It is important to plan several simultaneous examinations or tests, so that sequential steps are kept to a minimum.
The first step is to make sure that the culture is pure. A tangled mass of filaments can easily carry a contaminant with it. This is best done on a rich medium, streaking to obtain single colonies. Inoculate pure cultures on a transparent minimal agar in a cross-hatch pattern.
Figure 6.1: Cross-hatch streak plate. View directly under microscope fitted with a long working distance objective (x25 and x40). Mature hyphae with spores should be looked for in the angles of the streaks.
Table 6.1: Appearance of pathogenic actinobacteria in exudates or tissue smears/sections
Genus / Appearance in exudates (pus) and tissueStreptomyces / Granules are rare, but when present are small (25-150um), white to yellowish, lobated, sometimes clubbed. Colonies form a loose mycelium consisting of Gram-positive and partially acid-fast branching filaments, which often fragment into coccoid elements. With Gram’s stain the filaments often stain irregularly giving a beaded appearance. Does not stain with H & E stain. /
Actinomyces / Pathogenic strains form yellow, brown or black granules 1 - 2 mm in diameter, which stain with H & E, often breaking up into parallel bands. Branched filaments do not fragment. Aerial mycelium characterized by medium to long chains of conidia, often in spirals or whorls, which are sometimes acid-fast.
A mixture of small cocci, rods and pear-shaped, Gram-positive bacteria. /
Rhodococcus / No granules are present. Diphtheroids (Gram-positive rods, with traces of branching). The rods often break up into coccoid cells. No aerial mycelium present. /
Dermatophilus / Organism commonly found on the under surface of skin crusts, where it is typically seen as irregular branched filaments which divide both longitudinally and transversely forming packets of coccoid cells. They are not acid-fast. Best stain is Giemsa, but Gram’s stain is satisfactory. /
Table 6.2: Gram-positive bacteria showing slight branching
TEST / Actinomyces / Rothia / Arachia / Propionibacterium / Bifidobacterium / Mycobacterium / Rhodococcus / Agromyces / Oerskovia / ArcanobacteriumAerobic (A)/ Facultative (F) Anaerobic (An) / F / F / F / F / An/CO2 / A / A / A / F / F
Anaerobic, better growth / + / - / + / + / + / - / - / - / - / -
Catalase / -/(+) / + / - / +/(-) / - / D / + / - / + / -
Acid-fast / - / - / - / - / - / + / (+) / - / - / -
Gelatine / -/(+) / d / d / +/(-) / - / - / + / D
Motile / - / - / - / - / - / - / - / - / D / -
Penicillin / S / S / S / S / S / R / S / R / S / S
Pigment / - / - / - / d / - / D / + / (+) / + / -
Nitrate / +/(-) / + / + / +/(-) / - / D / d / - / + / -
Urea / d / - / d / D / d / - / -
O/F glucose / + / + / + / + / + / D / - / d / + / +
Aesculin / d / + / - / d / d / + / + / -
Starch / d / - / -/(+) / - / d / - / + / D
Casein / -(d) / - / - / - / - / - / + / D
Sabouraud’s dextrose / - / - / -
Growth at 55°C / - / - / - / - / - / d / - / - / - / -
ONPG / - / - / d / - / + / d
Set several sterile coverslips into the agar at angles so that they can be examined at different times without disturbing the growth near the others (Figure 6.2).
Figure 6.2: Inclined coverslips for observing actinobacterial morphology. Inoculate agar plate with coverslips inserted at an angle. After incubation withdraw coverslips and mount, upper surface down, in a water-containing wetting agent.
Alternatively, agar blocks on sterile slides covered by sterile coverslips may be inoculated on the sides of the blocks. The slide is placed in a moist chamber and incubated. It can be regularly examined microscopically without disturbing the growth (Figure 6.3).
A lipid-rich medium such as egg yolk agar, milk, serum or glycerol is also inoculated for acid-fast staining. Thioglycollate with gelatine (5%) is useful for determining whether the culture grows well anaerobically and determines motility and gelatine liquefaction as well. An antibiogram differentiates between members of this group and fungi. Penicillin is included as a distinguishing test.
Figure 6.3: Slide culture. Thin agar block, cut from poured plate, is placed on a sterile microscope slide and inoculated, and a sterile coverslip is applied. After incubation in a moist chamber, view slide culture directly on microscope stage when it should be possible to see the aerial mycelium (A) and the substrate mycelium (B) within the agar.
Table 6.3: Gram-positive bacteria showing extensive branching (A= aerobic, F= facultative anaerobe)
TEST / Nocardia / Streptomyces / Dermatophilus / Actinomadura / Nocardiopsis / Thermomonospora / Saccharomonospora / SaccharopolysporaAerobic (A)/ Facultative (F) / A / A / F / A / A / A / A / A
Anaerobic, better growth / - / - / - / - / - / - / - / -
Catalase / + / + / + / + / + / + / + / +
Acid-fast / (+) / - (Spores +) / - / - / - / - / - / -
Gelatine / d / + / + / + / + / + / +
Motile / - / - / + (spores) / - / - / - / - / -
Penicillin / R / R / S / R / R / S / S / R
Pigment / d / d / + / + / + / + / + / +
Nitrate / +/(-) / + / - / + / d / - / - / d
Urea / +/(-) / d / + / d / + / d
O/F glucose / + / - / + / +
Aesculin / + / + / + / + / +
Starch / d / + / + / d / + / + / d
Casein / -/(+) / + / + / + / + / + / + / +
Sabouraud’s dextrose / + / + / - / + / + / + / +
Growth at 55°C / - / - / - / + / - / + / + / +
ONPG / + / + / - / - / + / - / - / -
Examine the colonies regularly microscopically (once or twice a week if slow-growing, daily if rapid growing). Either place the agar block slide under the microscope and examine under high dry power without staining, or remove one of the cover slips angled in the agar. The growth adhering to the cover slip may be stained if required. Some Actinomyces spp. only branch at a very early stage (spider colonies) when still so small that they are not visible to the naked eye.
Note any branching, whether most of the branches are right angled or oblique, and whether all the growth is on or in the agar, or whether an aerial mycelium is formed (Figs. 6.4 and 6.5).
Figure 6.4: Streptomyces, Actinomadura, Actinomyces and Nocardia: coccoid, diphteroid fragments of filaments; aerial mycelium fragments to form unsheathed arthrospore-like units.
Figure 6.5: Agromyces, Dermatophilus, Actinomyces and Rhodococcus species have a rapidly fragmenting substrate mycelium; the segments become rounded and the colony usually consists of a mass of coccoid elements.
This is first and best seen in the angles of growth on the cross-hatched plate. Members of this group can undergo a rod-coccus or rod-coccus-hyphus cycle which means that they are usually seen as cocci during the resting stage.
Once placed on fresh medium, outgrowths occur so that they resemble rods which may develop into rudimentary hyphae which fragment again. When nutrients are depleted, cocci are again formed. The rate of growth as well as the individual isolate determines at which stage each change occurs. As this rate is difficult to predict, regular examinations will show changes as they occur. Aerial mycelia or filaments are darker and more retractile than those growing in the agar and may bear spores, the shape, number and size of which should be noted. As soon as growth is noted on the lipid rich medium, modified acid fast staining can be done as well as the catalase test.
These few tests should either be sufficient to identify the isolate or indicate whether it is a potential pathogen or contaminant. Further tests which are helpful in this group are nitrate, urease, acid production on oxidation and fermentation medium, aesculin, starch or casein hydrolysis, growth on Sabouraud's agar, growth at 55°C and ONP.