Principles and Practice of Clinical Bacteriology Second Edition - Free

30 ORAL AND OTHER NON-β-HAEMOLYTIC STREPTOCOCCI
process has reached the periapical region, infection either may remain
localized as an acute dental abscess or as a chronic granuloma or may
spread more widely in various directions depending on its anatomical
position. In some cases such spreading infections may cause a lifethreatening
situation, for example, if the airway is obstructed by
submandibular swelling (Ludwig’s angina).
The disease is initiated by acid demineralization of the teeth
because of the metabolic activities of saccharolytic bacteria, including
streptococci, which are situated on the tooth surface as part of the
complex microbial community known as dental plaque. Dental plaque
accumulates rapidly on exposed tooth surfaces in the mouth and
consists of a complex mixture of bacteria and their products. Many of
the oral streptococci listed in Table 2.1 are prominent components of
dental plaque. When an external source of carbohydrate becomes
available, in the form of dietary carbohydrate (particularly sucrose),
streptococci and other plaque bacteria rapidly utilize the fermentable
sugars and release acidic metabolic end products such as lactic acid.
This can result in a rapid drop in pH in the vicinity of teeth that, if
sufficiently low (pH 5.5 or less), in turn results in the demineralization
of the dental enamel.
Most detailed studies on the pathogenesis of dental caries have
focused on the mutans streptococci since these are widely regarded as
the most significant initiators of the disease. Properties of these streptococci
that are considered to be important in caries include ability to
survive and grow at relatively low pH, production of extracellular
polysaccharides from glucose and production of acid from carbohydrates,
and mutant strains lacking in one or more of these attributes
have been shown to be less cariogenic in experimental animals
(reviewed by Kuramitsu, 2003). The frequent consumption of sugar is
proposed to shift the plaque population in favour of aciduric species
able to grow and survive in low pH conditions, which in turn results in
greater plaque acidification and, consequently, greater enamel dissolution
(Marsh, 2003). However, oral species other than mutans group
streptococci are thought to contribute to the disease process.
The main approaches to caries prevention include the control of
dietary carbohydrates, particularly by reducing the frequency of sugar
intakes, the use of fluorides (both topically and systemically), maintenance
of good oral hygiene and plaque control, application of fissure
sealants and regular dental check-ups. With a better understanding of
mucosal immunity and using new technologies available in molecular
biology, researchers have developed novel caries preventative
measures. These include local passive and active immunization,
replacement therapy (the use of engineered noncariogenic S. mutans
strains to replace cariogenic species within dental plaque) and the use
of anti-adhesive peptides (Kelly et al., 1999; Hillman, 2002; Koga
et al., 2002). Despite growing evidence from laboratory animal and
human clinical studies of the ability of these approaches to control
S. mutans numbers, their potential as anti-caries treatments has yet to
be fully realized.
LABORATORY DIAGNOSIS
Specimens and Growth Media
Non-β-haemolytic streptococci are isolated from a wide range of
clinical specimens such as blood, pus, wounds, skin swabs and biopsies
as well as from dental plaque, saliva and other oral sites. Obtaining
pus from an oral abscess is best done by direct aspiration using a
hypodermic syringe rather than by swabbing, to reduce the risk of
contaminating the sample with the oral flora, although in some
instances (e.g. with infants), swab samples may be the only option
available, unless the patient is undergoing a general anaesthesia.
When sampling oral sites for ecological studies, it is necessary to
ensure that the area sampled is small enough to be representative of
a discrete site to avoid the risk of obscuring the differences between
sites by sampling too big an area.
Where the laboratory processing of a specimen may be delayed, the
clinical sample is best held in a suitable reduced transport fluid such
as the one described by Hardie and Whiley (1992).
The non-β-haemolytic streptococci are fastidious organisms, with a
need for a carbohydrate source, amino acids, peptides and proteins, fatty
acids, vitamins, purines and pyrimidines. These bacteria therefore need
complex growth media commonly containing meat extract, peptone and
blood or serum. Non-β-haemolytic streptococci are best isolated from
clinical samples on a combination of nonselective and selective agar
media. Nonselective examples include blood agar 2 (Oxoid, Hampshire,
UK), Columbia agar (Gibco BRL, Life Technologies, Paisley, UK),
fastidious anaerobic agar (Laboratory M, Amersham, UK) and brain–
heart infusion agar (Oxoid) supplemented with 5% defibrinated horse or
sheep blood. Several selective media are available for the non-β-haemolytic
streptococci. The two most commonly used agars are trypticase–yeast–
cystine (TYC) and mitis–salivarius (MS) agars that contain 5% sucrose
to promote the production of extracellular polysaccharides, resulting in
the production of characteristic colonial morphologies as an aid to identification.
TYC is available commercially from Laboratory M; MS agar
is available from Oxoid and from Difco (Detroit, MI, USA). Other
selective media commonly used for the isolation of mutans streptococci
are based on TYC or MS agars and have the addition of bacitracin
(0.1–0.2 U/ml) and an increased amount of sucrose (20%). Nalidixic
acid–sulphamethazine (NAS) agar is a selective medium for the anginosus
group streptococci and uses 40 g/l of sensitivity agar supplemented with
30 μg/ml of nalidixic acid, 1 mg/ml of sulphamethazine (4-amino-N-
[4,6-dimethyl-2-pyrimidinyl]benzene sulphonamide) and 5% defibrinated
horse blood. This medium is also selective for S. mutans.
As streptococci are facultative anaerobes, incubation is best carried
out routinely in an atmosphere of air plus 10% carbon dioxide or in an
anaerobic gas mix containing nitrogen (70–80%), hydrogen (10–20%)
and carbon dioxide (10–20%). Some strains have an absolute requirement
for carbon dioxide, particularly on initial isolation. Colonies on blood
agar are typically 1 mm or less in diameter after incubation for 24 h at
37 °C, are nonpigmented and often appear translucent. On blood agar
the streptococci discussed in this chapter usually produce α-haemolysis
or are non-(γ)-haemolytic. However, as mentioned previously, the
haemolytic reactions of different strains within a species may vary and
are sometimes influenced by the source of blood (e.g. horse, sheep) and
by the incubation conditions. Some examples of colonial morphology
on different culture media are illustrated in Figure 2.4.
Liquid culture of these streptococci may be carried out in a
commercial broth such as Todd–Hewitt broth or brain–heart infusion
broth (Oxoid) with or without supplementation with yeast extract
(0.5%). The growth obtained in broth cultures varies from a diffuse
turbidity to a granular appearance with clear supernatant depending on
strain and species.
Initial Screening Tests
Streptococci are usually spherical, with cells of approximately 1-μm
diameter arranged in chains or pairs. The length of the chains may vary
from only a few cells to over 50 cells, depending on the strain and
cultural conditions (longer chains are produced when the organisms are
grown in broth culture). Streptococci stain positive in the Gram stain,
although older cultures may appear Gram variable. Some strains may
appear as short rods under certain cultural conditions. Isolates should
be tested for catalase reaction, and only catalase-negative strains
should be put through further streptococcal identification tests.
The clinical microbiologist should be aware that, because of developments
in taxonomic studies, several other genera of facultative
anaerobic Gram-positive cocci that grow in pairs or chains have been
proposed that may superficially resemble streptococci. The scheme
described in Table 2.8 should allow the differentiation of these genera
on the basis of a few cultural and biochemical tests, with great caution
to be exercised in the interpretation of morphological observations.