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Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteria

Chapter 6: Aerobic Gram-positive filamentous bacteria

Applied Veterinary Bacteriology and Mycology: Identification of aerobic and facultative anaerobic bacteriaChapter 6: Aerobic Gram-positive filamentous bacteria

Author: Dr. M.M. Henton

Licensed under a Creative Commons Attribution license.

TABLE OF CONTENTSTable 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

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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

INTRODUCTIONThe 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

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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 tissue

Streptomyces 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.

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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

Act

inom

yces

Rot

hia

Ara

chia

Prop

ioni

bact

eriu

m

Bifi

doba

cter

ium

Myc

obac

teriu

m

Rho

doco

ccus

Agr

omyc

es

Oer

skov

ia

Arc

anob

acte

rium

Aerobic (A)/ Facultative (F) Anaerobic (An) F F F F An/

CO2A A A F F

Anaerobic, better growth + - + + + - - - - -Catalase -/(+) + - +/(-) - D + - + -Acid-fast - - - - - + (+) - - -Gelatine -/(+) d d +/(-) - - + DMotile - - - - - - - - D -Penicillin S S S S S R S R S SPigment - - - d - D + (+) + -Nitrate +/(-) + + +/(-) - D d - + -Urea d - d D d - -O/F glucose + + + + + D - d + +

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Aesculin d + - d d + + -Starch d - -/(+) - d - + DCasein -(d) - - - - - + DSabouraud’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.

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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.

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Table 6.3: Gram-positive bacteria showing extensive branching (A= aerobic, F= facultative anaerobe)

TEST

Noc

ardi

a

Stre

ptom

yces

Der

mat

ophi

lus

Act

inom

adur

a

Noc

ardi

opsi

s

Ther

mom

onos

pora

Sacc

haro

mon

ospo

ra

Sacc

haro

poly

spor

a

Aerobic (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).

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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.

Dermatophilus Congolensis

Dermatophilus congolensis is the agent of cutaneous streptothricosis and Sencobo disease in cattle and lumpy wool and strawberry foot rot in sheep. Other domestic animals and humans may also be affected.

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The disease is important in countries in southern, central and West Africa, the Caribbean and Israel. The infection is characterized by the formation of thick crusts which come away with a tuft of hair, leaving a moist depressed area with bleeding points from capillaries. Morbidity can be high, but mortality is low. The position of the lesions in the skin varies with predisposing conditions such as water maceration and heavy infestation with Ambylomma ticks.

Laboratory diagnosis

1. Specimen collection

Scabs, biopsies or exudates collected from unopened pustules are best. Exudates should be submitted to the laboratory in sterile tubes or on swabs moistened with sterile water. Biopsy specimens should be submitted in sterile screw cap bottles without preservative. The samples should be refrigerated if culturing is to be delayed. Scabs, scrapings and crusts may be transported dry, in sterile tubes or jars and stored at ambient temperatures.

2. Direct microscopy of smears

Actively growing organisms are found on the undersides of the scab and in the epidermis at the edge of the lesion. Collect scabs from different sites because the organisms may be scanty and difficult to demonstrate. D. congolensis survives for months, even years, in dry scabs but is soon killed in wet scabs by contaminating bacteria. Make a thick smear from the underside of the scab, if necessary soaking in a bit of distilled water or saline. Stain with 10% Giemsa for 30 minutes, DiffQuik or methylene blue (1% for 30 seconds). Although Gram’s stain can be used, both the cells of D. congolensis and debris absorb the crystal violet-iodine stain avidly staining too darkly. A modification is to leave the crystal violet on the smear for only 2-3 seconds, after which the morphology of the bacterium is easier to see. Giemsa and Grocott methenamine-silver stains are best for paraffin sections, which often reveal the micro-organism abundantly in hair follicles, accompanied by large numbers of eosinophilic leukocytes.

The appearance of D. congolensis is unique, in that a presumptive diagnosis can be made by the examination of glass smears by light microscopy. A search must be made for the typical chains of blue cocci (zoospores) in two, four or eight parallel rows resulting in a “tram-track” or “cat’s paws” appearance. Single chains or single cocci resemble other common cocci and cannot be distinguished from them. Typical chains can be hard to find even from lesions which look active and it is advisable to search several smears before declaring them negative. If the flakes of scab are treated too roughly when the smears are made, the filaments will disintegrate and only Gram-positive cocci (zoospores) will be seen. Table 6.1 and Fig 6.6 show the morphology of D. congolensis.

3. Culture

Uncontaminated specimens can be directly streaked onto appropriate media. It is best to use the underside of scabs or crusts or exudates. As scab material often contains many contaminants, Haalstra’s method for the primary isolation of D. congolensis is recommended:

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1. Make a suspension of scab material by grinding with sterile sand and distilled water. Place the material in 2ml of distilled water in a sterile bijou bottle (or any other suitable bottle) for 3.5 hours at room temperature.

2. Place the container with lid removed, in a candle jar at room temperature for 15 minutes.3. The motile zoospores are chemotactically attracted to the carbon dioxide-enhanced atmosphere in

the candle jar and move to the surface of the distilled water.4. Remove a loop full of fluid from the surface and streak on to a blood agar plate. 5. Incubate in a candle jar at 37°C for 48 to 72 hours. Occasionally five days are required for colony

formation.

The blood agar plate often shows a mixed growth of skin bacteria with a few D. congolensis colonies. Colonies are very characteristic, white or yellow (2 - 5 days), raised with an irregular rough surface (confluent growth may appear mucoid). In addition, they show a narrow zone of beta-haemolysis, pit the medium and are hard to lift off the surface. No growth occurs on Sabouraud’s dextrose agar. The beta-haemolysis is best seen on horse blood agar. These bacteria unlike Nocardia species will grow anaerobically. Dermatophilus congolensis colonies often produce short aerial hyphae when cultured in a candle-jar.

Microscopic appearance

Smears from colonies do not show the characteristic “tram-track” appearance seen on direct microscopy. Gram’s stain reveals a mycelium of Gram-positive branching filaments, some of which are broken down transversely and longitudinally into chains of cocci i.e. parallel rows of cocci. If only cocci are seen and D. congolensis is suspected, cultures should be examined at an earlier stage for hyphae.

Biochemical reactions

As the colony morphology and microscopic appearance is characteristic, biochemical tests are usually not performed. D. congolensis is catalase negative, urease negative and gelatin positive and produces weak acid from glucose, fructose and maltose. It is indole negative, does not reduce nitrate and does not attack sucrose, salicin, xylose, lactose, sorbitol, mannitol or dulcitol. It hydrolyzes starch but not xanthine or tyrosine.

4. Serological diagnosis

A highly specific immuno-diffusion test has been developed to detect circulating antibodies in chronically infected cattle but is not used as a routine diagnostic test.

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Fig. 6.6: Developmental cycle of Dermatophilus congolensis

Nocardia

Nocardia infections occur sporadically in domestic and wild animals. They are important because lesions resemble those of tuberculosis and because they cause tuberculin sensitivity. Nocardia asteroides is a cause of a purulent thoracic effusion and deep-seated abscesses in cats and dogs and abortions, jaw abscesses and mastitis in cattle. In other animals various purulent infections are found. Safety precautions used for Mycobacterium infections should be followed when handling samples.

The nocardiae are aerobic, catalase-positive, Gram-positive, non-motile, branching filamentous bacteria that fragment into irregularly shaped rods and cocci. Aerial hyphae are produced. With the exception of N. amarae, all nocardiae are resistant to lysozyme. As the cell wall contains mycolic acids, these bacteria often stain partially acid-fast. The taxonomy of Nocardia is rapidly changing at present.

Laboratory diagnosis

1. Specimens

Specimens should include exudates, aspirates, mastitis milk samples, tissue from granulomas and thin sections from granulomas in 10% formalin for histopathology. Swabs are not recommended as the transport media may make identification by smear examination difficult. If swabs must be used they should rather be moistened with a little sterile water. Samples suspected of containing Nocardia should not be refrigerated or placed on ice prior to being inoculated onto appropriate culture media. Some strains of N. asteroides and N. brasiliensis rapidly lose viability when exposed to near freezing temperatures. Blood cultures may be taken if disseminated disease is suspected. These cultures should however be incubated for at least two weeks and are often unrewarding.

2. Direct microscopy

Soft “sulphur” granules (Splendore-Hoepli phenomenon) are not common in exudates from N. asteroides infections. Gram - and MZN-stained smears should be made from exudates, aspirates, granulomatous tissue and centrifuged deposits of bovine mastitis milk. As the cell wall of Nocardia species contains mycolic acids of intermediate size, they are able to retain carbol fuchsin when rinsed with a weak mineral acid, then 70% of Nocardia isolates will stain partially acid-fast. Acid-fast staining is best seen when

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cultured on a lipid-rich medium such as egg yolk or milk. Nocardiae are usually not apparent in haematoxylin and eosin stained sections and are best visualized by the Brown and Brenn modification of the Gram’s stain.

Nocardia appear as a mycelium of delicate, branching filaments in which staining may be uneven. The filaments often show fragmentation into coccobacillary elements. They can resemble Mycobacterium spp. (acid-fast stain) and Corynebacterium spp. (Gram’s stain). They are found both intra- and extracellularly. They do not form Gram-negative clubs, which differentiates them from Actinomyces.

3. Culture

Nocardia will not survive the decontamination techniques used to isolate mycobacteria, but they will grow on blood agar and Sabouraud’s dextrose agar (Oxoid). Inoculate pus and tissue suspensions on blood agar and Sabouraud, and incubate aerobically at 37°C. For contaminated samples paraffin agar or buffered charcoal agar used for the isolation of Legionella are effective. Even though Nocardia are seen on direct smears, they can be problematic to culture and thus plates should be kept for at least two weeks before being discarded. During this period, the plates should be kept well sealed in order to maintain a moist environment. This period of extended incubation can also result in the overgrowth of contaminating bacteria or fungi, which may hide or inhibit the growth of nocardiae. Furthermore, nocardiae may manifest extremely variable morphologies on different culture media, which may make recognition difficult. In fact macroscopically, the colonies of Nocardia and Mycobacterium spp. can look the same on media designed for the cultivation of Mycobacterium spp. Examination of colonies under a stereo microscope can help one recognize the growth characteristic of nocardiae.

Some Nocardia may become physiologically and structurally altered as they grow within host tissues, with the result that transferral of these altered organisms may be lethal for them or requires an extended period to adapt the organisms to the new environment.

Colonies appear after three to five days, raised, white, pink or yellow, smooth or granular, irregularly folded (cauliflower-like). On sheep or bovine blood agar, many clinical isolates of N. otitidiscaviarum, N. brasiliensis and rarely N. asteroides initially are small with a narrow zone of alpha-haemolysis around the colony. After 2 - 7 days, the colony widens, digs into the agar and often has a zone of beta-haemolysis around it. However, some isolates of Nocardia spp. exhibit only alpha haemolysis and most isolates of N. asteroides appear to be non-haemolytic on sheep blood agar. On Sabouraud’s agar colonies are orange, glabrous, heaped and folded to white or pink, raised and chalky with aerial hyphae.

Stained smears from growth on laboratory media will show coccoid and bacillary elements. The long, branched filaments are usually not seen from agar preparations but are easily obtained from growth in broth. Acid-fastness is best seen when the organism is cultured on a high lipid media such as litmus milk or Løwenstein-Jenssen media. On Sabouraud’s dextrose agar, arthroconidia-like elements occasionally form aerial hyphae.

4. Identification

These bacteria are difficult to distinguish from Streptomyces species. Identification of the genus is based on partial acid-fastness, inability to grow anaerobically, the production of aerial hyphae and the ability to

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grow on Sabouraurd’s dextrose agar. Nocardia asteroides often forms a brown pigment on Sabouraud’s dextrose agar. Species identification is more difficult and should be referred to a reference laboratory.

Tentative species identification can be performed by mycolic acid profiles using gas chromatography and HPLC analysis. These tests are however difficult to standardize due to the great number of variables. The use of DNA probes for the identification of the Nocardia is likely to enhance identification in future.

5. Antimicrobial susceptibilities

Presently there are no standardized methods of susceptibility testing for Nocardia adopted by the NCCLS, as it is difficult to make a uniform suspension of these bacteria for the test and the fact that these organisms are slow-growing. However the most common methods presently employed are the disk-diffusion method using Mueller-Hinton agar and a broth micro dilution method using Mueller-Hinton broth. The E-test has also proven to be useful. Antimicrobials used in the panel should include sulfamethoxazole in combination with trimethoprim, amoxycillin-clavulanic acid, tetracycline, amikacin, ceftriaxone, cefotaxime and imipenem.

6. Serology

Reliable immunodiffusion tests have been established for systemic nocardiosis in dogs and nocardial mastitis in cattle, but are not routinely used. In humans, an indirect immunofluorescence test is used to screen patients. However because of the cross-reaction of the test with Mycobacterium spp. and other actinobacteria, samples for culture should be taken from patients with positive test results.

7. Interpretation and reporting of results

As Nocardia are common in the environment, but rare in clinical specimens, results should be interpreted with consideration of clinical disease, and the presence of bacteria with typical Nocardia morphology on exudates or tissue smears. Due to the fact that it is not always possible to isolate Nocardia spp. from clinical specimens, a presumptive result can be reported if Gram-positive or partially acid-fast staining branching filamentous bacteria that broke up into coccoid elements were seen in tissue or exudate smears, particularly if the organism stained acid-fast.

Table 6.4: Hydrolysis and selected biochemical profiles of pathogenic species of Nocardia

Decomposition of: Growth Arylsulfatase Acid from

Organism

Cas

ein

Hyp

oxan

thin

e

Tyro

sine

Xant

hine

Gel

atin

Star

ch

at 4

5°C

(14

day)

Glu

cose

Rha

mno

se

N. asteroides - - - - - - +/- - + -/+N. farcinica - - - - - - + - + +/-N. nova - - - - - - - + + -N. brasiliensis + + + - + - - - + -

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N. otitidiscaviarum - + - + - - +/- - + -N. transvalensis - +/- - +\- - + - - + -Streptomyces spp. + - + + -

Table 6.5: Differentiation of the causative agents of canine nocardiosis and canine actinomycosis

Canine nocardiosis Canine actinomycesAetiology Nocardia asteroides Actinomyces viscosusGranules in exudates Not common Usually presentFilaments MZN-positive + (70%) -Fragmentation of filaments + -Growth on Sabouraud’s dextrose agar + -Powdery, white colonies (aerial hyphae) + -Susceptibility to penicillin - +Anaerobic growth - +

Streptomyces

The genus Streptomyces consists of numerous poorly defined species, which are aerobic, catalase positive, Gram-positive, non-motile, extensively branched filamentous bacteria that form aerial hyphae, with short to long chains of non-motile spores. Over 3 000 species of Streptomyces have been identified. These soil saprophytes are rarely implicated in clinical disease, but are common laboratory contaminants. Because they are similar in colony and microscopic appearance to Nocardia and sometimes Actinomyces species they should be distinguished from them. Streptomyces spp. tends to form colonies with a white powdery surface that emit a pungent musty-basement odour. The branching filaments usually do not break up and the aerial hyphae produce conidia that are typically chained. Streptomyces species, unlike Nocardia, do not stain acid-fast. They are also sensitive to lysozyme and rapidly hydrolyze casein.

Actinomyces and Arcanobacterium

Actinomyces are Gram-positive, diphteroidal or branching filamentous rods. They are anaerobic or microaerophilic commensal organisms found in the oral cavities of animals and humans. In this section only Actinomyces spp. which grows under aerobic conditions will be discussed. Actinomyces viscosus is the cause of localized or granulomatous abscesses or pyothorax in canines.

Arcanobacterium pyogenes was formerly called Actinomyces pyogenes and previously Corynebacterium pyogenes

Diagnosis

Specimen collection

Specimens include pus, exudates, aspirates, tissue and scrapings from the walls of abscesses if they have been incised. A volume of fluid or pus should be collected and submitted, if possible, rather than just a small amount on a swab. Thin sections of granulomas in 10% formalin are useful for histopathology.

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Smear examination

“Sulphur” granules (Splendore-Hoepli bodies) are the best specimens for the visualisation of A. viscosus. The pus or exudate is placed in a Petri dish and washed carefully with a little distilled water to expose the soft greyish-white granules of A. viscosus. A granule is placed on a microscope slide in a drop of 10% KOH and gently crushed by applying pressure on the coverslip. The characteristic clubs can be seen if the preparation is examined under the low power objective of a microscope. To visualise the bacteria, a Gram’s stain is made, which reveals delicate, Gram-positive, branching filaments. Occasionally short filaments or pleomorphic diptheroidal forms may predominate.

Isolation

Material is inoculated onto blood agar plates and incubated in air with 5 – 10% CO2. (Or candle jar). Actinomyces viscosus will usually grow in 2 - 4 days.

A. viscosus usually produces two colony forms: one is smooth, entire, convex, glistening, mucoid or soft colony composed of diptheroidal forms; and the other consists of rough, irregular, heaped, granular and slightly dry colonies, which yield branching filaments. Both forms are usually seen and neither is haemolytic. It is distinguished from other Actinomyces spp. in that it grows well aerobically, and from Nocardia or Streptomyces by its failure to grow on Sabouraud’s dextrose agar.

Biochemical tests

Table 6.6: Identification of Actinomyces spp. (usually glucose positive)

Spec

ies

Aer

o to

lera

nce

Cat

alas

e

Aes

culin

hyd

roly

sis

Gel

atin

hyd

roly

sis

Fermentation of

Ure

ase

Nitr

ate

redu

ctio

n

Mel

ezito

se

Ara

bino

se

Gly

coge

n

Man

nito

l

Raf

finos

e

Rib

ose

Salic

in

Sucr

ose

Treh

alos

e

Xylo

seA. bovis M or

An - - - - - - - - - - -

A. bowdenii F + + - - + + - - - + + + -A. canis F + - - - - - -+ + - + v - +A. catuli F v + - - + v - - + + + +

A israelii M or An - - - V V + + +

A. odontolyticus M or An - - - + - - V V

A. howellii F + + - - - v - - - + - - + v +A. naeslundi F (-) (+) - w + - (-) - + - V (+) + VA. viscosus* F + + - V V - - - + - V + v -A. pyogenes F - + - - - - - VA. hordeovulneris

M or An (+) + w - - - v v - w v + + v +

A. denticolens F - ? - + D -A. hyovaginalis F - + - - v - (+) -(d) + + - +A. neuii anitratus F + - - - - - - + + + + (+)A. neuii neuii F + - - - + - - + v + v +A. slackii F + + v (-) + - - - - - v + +A. suimastitidis F - + - - - (+) - + + + - +

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Spec

ies

Aer

o to

lera

nce

Cat

alas

e

Aes

culin

hyd

roly

sis

Gel

atin

hyd

roly

sis

Fermentation of

Ure

ase

Nitr

ate

redu

ctio

n

Mel

ezito

se

Ara

bino

se

Gly

coge

n

Man

nito

l

Raf

finos

e

Rib

ose

Salic

in

Sucr

ose

Treh

alos

e

Xylo

se

A. vaccimaxillae F - + - - - - + - - - + + +Actinobaculum suis An v - - - v - - +

* Serotype 1 does not produce pyrazinamidase but serotype II does.

Susceptibility to antimicrobial agents

Strains of Actinomyces are usually sensitive to a wide range of antimicrobials, and evidence of the development of resistance is lacking. However as Actinomyces spp. are usually components of polymicrobial infections, and other bacteria with antimicrobial resistance may protect the actinobacteria.

These bacteria are usually sensitive to penicillins and tetracycline and usually resistant to metronidazole, the aminoglycosides and peptide antibiotics.

Serological identification

Reference laboratories make use of serological identification to identify the different actinobacteria.

REFERENCES

1. The Actinomycetes. In: Clinical Veterinary Microbiology, Eds Quinn P J, Carter M E, Markey B and Carter G R (1994). Wolfe Publishing. 144 - 155.

2. Nocardioforms and aerobic actinomycetes. In: Colour Atlas and Textbook of Diagnostic Bacteriology (4th Edition). Eds. Koneman E W, Allen S D, Janda W M, Schreckenberger, & Winn W C (1992). J B Lippincott Company

3. Veterinary Microbiology and Microbial Disease, (2011). Quinn, P.J., Markey, B.K., Leonard, F.C., FitzPatrick, E.S., Fanning, S., Hartigan, P.J. Wiley-Blackwell. ISBN 978-1-4051-5823-7

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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

A. k

entu

ckye

nsis

A. l

exin

gton

ensi

s

A. p

reto

riens

is

A. m

edite

rran

ei

A. a

lba

A. a

lbid

ofla

vus

A. a

zure

a

A. c

olor

aden

sis

A. e

uryt

herm

a

A. f

astid

iosa

A. j

apon

A. m

etha

nolic

a

A. o

rient

alis

sub

sp.

orie

ntal

is

A. r

ubid

a

A. s

acch

ari

A. s

ulph

urea

A. t

herm

ofla

va

Decomposition of:Allantoin - - - - + + - - + - + + - - + - +Casein + + + + + + + + + + + - + + + + +Aesculin + + + + + + + + - - + w + + + + +Gelatin + + + + + + + + + - + + + w + + -Hypoxanthine + + + + + + + + w - + + + + - - +Starch - - - - + - - + - - + - + - - - -Urea + + w + + w + - + + - - + + + - +Xanthine - - - - + + - - - - + - + + + - +Production of:Soluble pigments + + + - - - + + - + - - - + - - +Nitrate reductase - + - - - + + + + + - + + + + + -Acid from:Adonitol + + - - + + + - + - + + + + + - +Arabinose + + + + + + + - + w + - + + + - +Cellobiose + + + + + + + + + + + + + + + - +Dextrin + + + + + - + + w w + - + - + + -Erythritol - w + - + + + - + - + + + + + - +Fructose + + + + + + + + + w + + + + + + +Galactose + + + + + + + + + w + + + + + + +Inositol + + + + + + + + + - + - + + - - -Lactose + + + + + + + - w - + - + - + - +Maltose + + + + + + + + - w + + + - + + -Mannitol - w - + + w + + + - + + + + + + +Melibiose + + + + + - + - - - + - - - - - +Methyl -D-glucoside + + + - + - + + - w + - + - + - +

Raffinose + + + + + - + - - w + - + - - - +Rhamnose + + + + - - - - + - - + + + + - -Salicin + + + + + - + + - - + w + + + - +Sorbitol + - w w - - - - + - - + - - - - +Sucrose + + + + + + + + - w + + + + + + -Trehalose + - + + w + + + + w + + + + + + +Xylose + + + + + + + + + - + + + + + - +Growth in/at: 5 % NaCl + + + w - + + + + - w + w + + - +

45 °C + + - - - - - - + + - + - - + - ++, Positive; -, negative; W, weak reaction

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APPENDIX 1Media and tests used in the identification of aerobic/aero-tolerant Gram-positive branching filamentous bacteria

1. Casein agar

Method

A

B

Skim milkDistilled water

AgarDistilled water

10g90ml

3g97ml

1. Autoclave A and B separately at 121°C for 10 minutes

2. Allow to cool to 50°C3. Combine and pour into Petri dishes

Interpretation

If casein is hydrolyzed a clear zone will develop around the inoculum

Positive: Streptomyces spp., N. braziliensis, T. pyogenesNegative: N. asteroides, N. otitidiscaviae, N. nova, N. farcinica

2. Tyrosine or xanthine agar

Method

Basal medium pH 7.0

Beef extractPeptoneAgarDistilled water

3g515g1 000ml

1. Heat to bring ingredients into solution.

2. Place 100ml aliquots into 250ml flasks. Sterilize by autoclaving at 121°C for 10 minutes

3. Allow medium to cool almost to solidification 4. Add 0,4g xanthine or 0,4g tyrosine to 100ml

of basal medium.5. Mix well and pour into Petri dishes.6. Incubate at room temperature for 4 weeks

and examine daily for clearing of medium below and around the colony.

Interpretation

Clearing below and around the colony indicates a positive result.

Xanthine

Positive: N. Otitidiscaviae and some Streptomyces spp.

Negative: N. asteroides, N. farcinica, N. Nova & N. brasiliensis

Tyrosine

Positive: N. brasiliensis & Streptomyces spp.

Negative: N. asteroides, N farcinica, N. Nova & N. Otitidiscaviae

3. Lysozyme broth

Method:

Glycerol broth (basal medium)

PeptoneGlycerolDistilled water

5g70ml1 000ml

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Divide into 2 equal aliquots and sterilize by autoclaving at 121°C for 15 min. One aliquot will serve as control. Lysozyme solution

Lysozyme0,01N HCl

100mg100ml

Sterilize by filtration.

1. To 95ml glycerol broth add 5ml lysozyme solution

2. Mix and tube aseptically in 5ml aliquots.3. All broths and lysozyme solution can be

stored in a refrigerator.

4. Place several fragments of culture into a tube containing lysozyme and one without lysozyme (control).

5. Set up a control set of broths by inoculating a tube with lysozyme and one without with Streptomyces spp.

6. Incubate all tubes at room temperature until control tubes show good growth, at which time the tests may be read and discarded.

Interpretation

Growth in lysozyme tube: Nocardia

No growth in lysozyme tube: Streptomyces

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