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 Isolation and identification of Rhodococcus
equi
 Isolation
The isolation of Rhodococcus
can be performed by plating the specimen onto blood agar medium. Incubate the
plates at 35oC in ambient air. On primary culture, the colonies are usually
visible after 24-48 hours and attain a diameter of 1-2 mm. Older colonies
tend to become larger and mucoid with a characteristic salmon-colored
pigment. Appearance of the pigment may be delayed.
Identification
Rhodococcus
can be presumptively identified via the staining technique. Any specimen that
shows gram-positive pleomorphic coccobacilli and stained red with modified
acid fast stain may be suspected as Rhodococcus.
The
characteristic colonies that are gram-positive pleomorphic coccobacilli or
diphtheroid-like should be subcultured. The pure culture of the suspected
organism can be identified by conventional biochemical tests.
The
modified acid fast staining from the colonies usually gives negative results.
Antimicrobial susceptibility test
According to the
NCCLS, there still have no standard zone interpretation using the Kirby-Bauer
susceptibility test for this organism. In cases of clinical importance, the
MIC technique may be performed where applicable. 
 Isolation
and identification of Salmonella
Isolation
The organisms belonging to
genus Salmonella are gram-negative, non acid fast, non capsulated and
non-sporing bacilli, which measure approximately 2-4 m m x 0.6 m m. Almost al
species of Salmonella are motile. The organisms grow rapidly on ordinary
media and optimum temperature of growth is 370C. The Salmonella spp. other
than S. typhi and S. paratyphi have been reported as
opportunistic bacterial infections in HIV/AIDS patients.
Identification
The growth of Salmonella on
MacConkey agar is pale yellow (non lactose fermenting) colonies, 2-3 mm in
diameter, moist, circular and smooth convex surface. These are catalase
positive and oxidase negative. The organisms can be identified by
conventional biochemical tests. An organism identified as Salmonella, on the
basis of biochemical reactions, can be confirmed with slide agglutination
test, using polyvalent ‘O’ and polyvalent ‘H’ antisera against salmonellae.
To perform this test, one drop each of normal saline is placed at two
different sites on a clean glass slide. A loopful of biochemically suspect
colony of Salmonella is emulsified in both. One of these is kept as control
and another is tested first with polyvalent ‘O’ antiserum against Salmonella.
If this gives visible agglutination within two minutes, the process is
repeated with polyvalent ‘H’ antiserum. If the serogroup is to be determined,
the test should be further performed with monovalent ‘O’ antisera against
Salmonella.
Antimicrobial susceptibility test
The emergence and
spread of Salmonella resistant to multiple antibiotics has assumed a massive
proportion. For this reason, conventional antibiotics such as
chloramphenicol, ampicillin and trimethoprim sulfamethoxazole can no longer
be considered as the first line drugs in the treatment. Each and every
isolate of Salmonella should be subjected to antimicrobial susceptibility
test using the standard method. This will not only help in the management of
the patient more efficiently but will also help in generating the
surveillance data which will be useful to formulate antibiotic policy in a
given hospital.
Nocardiosis
Nocardiosis
is an acute or chronic, suppurative (less commonly granulomatous) disease
caused by the soil-inhabiting aerobic actinomycetes, Nocardia asteroides,
Nocardia brasiliensis, and Nocardia otitidiscaviarum. There are three
distinct clinical syndromes that may evolve : (1)
primary cutaneous, (2) primary subcutaneous and (3) primary pulmonary and
systemic.
Natural habitat
Gordon
and Hagen first isolated N. asteroides from soil by the paraffin technique.
These findings have been confirmed by other investigators, and it appears
that the organism has a worldwide distribution. N. brasiliensis and N.
otitidiscaviarum have also been recovered from soil.
Several
species of the genus Nocardia, viz. N. asteroides, N. brasiliensis, N. caviae
and perhaps N. farcinica, are valid etiologic agents of the clinical disease
nocardiosis in humans. N. brasiliensis is more virulent than N. asteroides or
N. otitidiscaviarum. It is able to cause infection readily in experimental
animals as well as systemic disease in normal patients.
Clinical manifestation
Nocardiosis
has appeared in multiple case reports, particularly in relation to its
association with other diseases and with the use of antileukaemic drugs,
cytotoxins, immune depressants, corticosteroids, and as a complication of the
AIDS. The opportunistic nature of the infection is now emphasized and there
are approximately 1 000 cases each year in the United
States. N. asteroides accounts for about
90% of cases, N. brasiliensis for nearly 7 % and N. otitidiscaviarum for less
than 3 %.
With
rare exceptions, nocardiosis is a pulmonary disease of respiratory origin
that results from the inhalation of spores. Cerebral nocardiosis associated
with pulmonary disease is frequently reported (about 27 per cent of pulmonary
infection involves the central nervous system also), and there is conclusive
evidence of hematogenous spread. Primary or secondary lesions in the
gastrointestinal tract at a site of pre-existing mucosal ulceration and,
rarely, appendiceal involvement may result from ingestion of contaminated
material or sputa from infected lungs.
Essential laboratory investigations and
their interpretation
Microscopy
Sputum, pus, tissue material and so forth can be
examined for N. asteroides. The materials to be examined may be digested, then concentrated by centrifugation. Gram’s stain of the
material will show long, sinuous, branching, gram-positive filaments and
fragmented bacillary bodies. The branching tends to be at long intervals and
at right angles to the main axis of the mycelium. A modified acid-fast stain
will show beaded or fragmented acid-fast bacillary forms. This distinguishes
the organism from Actinomyces, but it may be confused with the tubercle
bacillus. Both N. asteroides and N. brasiliensis are acid-fast
; most other actinomycetes are not. Rarely, N. asteroides aggregates
into a soft pseudogranule, whereas N. brasiliensis regularly forms true
granules.
Culture
N. asteroides grows readily on
ordinary laboratory media without antibiotics. It is aerobic but it grows out
equally well under anaerobic conditions. Its optimal growth temperature is
370C. Both N. asteroides and N. brasiliensis develop slowly on routine media,
particularly on Sabouraud’s agar. By two to three weeks, they attain a
diameter of 5 to 10 mm. The colonies are waxy, folded and heaped at first.
They may later develop areas of downy or tufted aerial mycelia. The whole
surface may become dry and powdery. A musty, dirt
like odor is sometimes present. The colour range included orange, pink,
white, buff, brown, lavender and salmon.
Serological tests
Serological
procedures have been tried on sera from patients with nocardiosis, but no
consistency of response has been found. Circulating antibodies have been
reported in patients with nocardiosis, but their role in defense has not been
assessed. Complement fixing and agglutinating antibodies have been reported.
A fluorescent antibody technique for specific identification of N. asteroides
has been attempted, but sensitivity was only 20 per cent or above. At the
present time, there is no reliable diagnostic or prognostic serological
procedure for the disease.
Isolation
and identification of Nocardia
Isolation
Nocardia, a member of the
actinomycetes, is gram-positive branching filamentous, often with a bead
appearance. Nocardia spp. are able to grow on
routine laboratory media such as sheep blood agar. However, because Nocardia
spp. grow slowly and require a minimum of 48 to 72 hours of incubation, it
may be overgrown by other normal flora present in contaminated specimens. A
solid medium that uses paraffin as the sole source of carbon has been
effective for isolating Nocardia spp. from contaminated clinical specimens.
Nocardia spp. also grow well on Sabouraud dextrose
agar but are inhibited by chloramphenicol. Thus, chloramphenicol should not
be added into the medium. Although Nocardia spp. grow
at 350C, some strains grow well at 300C. Plates should be incubated for 2 to
3 weeks. The colonial appearance of Nocardia spp. are
extremely variable; some isolates are beta-hemolytic on sheep blood agar.
Some are wrinkled; often dry, chalky-white appearance to orange-tan pigment.
Identification
The Gram stain from suspected
colony should be performed. If Gram-stain morphology is suggestive of a
possible Nocardia spp. ie. Gram-positive branching, fine, delicate filaments
with fragmentation, a Ziehl-Neelsen acid-fast stain should first be
performed, followed by a modified acid-fast stain. If the modified acid-fast
stain results are positive, the isolate may be reported, preliminarily, as
Nocardia spp. If acid-fast stain-negative, these organisms are still not
completely ruled out because of the variability of acid-fastness among
isolates belonging to this group. To confirm the identification, the isolate
should be referred to a reference laboratory.
Antimicrobial
susceptibility test
Although various methods are available, the antimicrobial
susceptibility testing of Nocardia spp. remains problematic. Some of the
problems include the lack of standardized, validated methods, lack of
correlation of in vital susceptibility testing results with clinical outcome,
and the inability to achieve a uniform suspension of organisms for testing
for all strains. Nevertheless, antimicrobial susceptibility testing should be
performed on clinically significant isolates and also should be referred to a
reference laboratory.
References
Forbes
BA, Sahm DF, Weissfield AS (1998) Diagnostic Microbiology, Tenth edition.
Beaman
BL, Saubolle MA, and Wallance RJ. (1995) Nocardia, Rhodococcus, Streptomyces,
Oerskovia, and Other Aerobic Actinomycetes of Medical Importance. p. 379-399.
In Murray PR, Baron EJ, Pfaller
MA, Tenover FC, and Yolken RH. (ed.) Manual of Clinical Microbiology Sixth
edition. American Society for Microbiology, Washington.
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