Fukuda et al.

Asian Pac J Dent 2012; 12: 37-44.

Tryptic Soy agar supplemented with bacitracin represents aremarkable advancement as a selective culture medium for
Streptococcus anginosus
Yu Fukuda, DDS,a Susumu Imai, PhD,b Yoshiki Hamada DDS, PhD,a and Nobuhiro
Hanada, DDS, PhDbab

Department of Oral and Maxillofacial Surgery, and Department of Translational Research, Tsurumi
University School of Dental Medicine, Yokohama, Japan
Purpose: To develop a new selective culture medium specifically for Streptococcus anginosus.
Materials and Methods: Three antibiotics, sulphamethazine (s), aztreonam (a), and bacitracin (b), were tested
by adding to Tryptic Soy (TS) agar at different concentrations. Initially, sulphamethazine (1.0 mg/mL) andaztreonam (0.2 mg/mL) were added to TS agar to obtain TS-sa agar. In total, 24 strains of oral bacteria
including three clinical S. anginosus isolates were used during the entire study. A minimal inhibitory
concentration (MIC) test was performed by using 96-well plate to determine the optimal bacitracin concentration
for the selective medium for S. anginosus under anaerobic condition at 37ºC for 48 hours. Finally, bacitracin
was added at a concentration of 2.0 U/mL to TS-sa agar to prepare TS-sab agar medium and selectivity wastested using all 24 strains of bacteria.
Results: Most strains of S. anginosus could grow on the TS-sab agar medium but other bacteria except for S.
intermedius and S. sobrinus could not be detected on this medium. S. intermedius and S. sobrinus occasionally
showed some growth on TS-sab agar medium with low detection rate. Although some clinical isolates of S.
anginosus were not enriched like other strains of S. anginosus, a remarkable enhancement was achieved in thisstudy in the development of a selective culture medium for S. anginosus.
Conclusion: TS-sab may serve as a remarkably improved semi-selective culture medium for S. anginosus.
Continued studies are required to improve the selectivity specific for S. anginosus.
(Asian Pac J Dent 2012; 12: 37-44.)
Key Words: aztreonam, bacitracin, selective medium, Streptococcus anginosus, sulphamethazine

Introduction
Streptococcus anginosus (S. anginosus) group bacteria are classified as the oral viridians streptococci and canbe isolated from several parts of the human body, including the oral cavity and gastrointestinal and genitourinary
tracts.1-4 It forms part of the normal flora of the human oral cavity and upper respiratory, gastrointestinal, andfemale urogenital tracts.5 S. anginosus is frequently isolated from purulent infections of the mouth and internalorgans, including the brain, liver, lungs, and spleen,1-8 and from cases of appendicitis, peritonitis, endocarditis,
meningitis, obstetric and neonatal infections, and infections of the skin and soft tissues.9 Although the organism
is generally considered to have a low pathogenicity, evidence for the pathogenicity of these streptococci has
recently been extensively reviewed9 and indicate that it has a propensity to cause severe purulent infections at
virtually all body sites. Notably, S. anginosus is the most common cause of brain and liver abscesses andpulmonary empyema.5,10,11 Furthermore, it has been reported that S. anginosus is associated with esophageal,
gastric, and pharyngeal cancer tissues.10,12-15
It is an important issue to develop a proper selective meium for these streptococci, due to the unusual
characteristics and unclear involvement of S. anginosus in human infectious diseases. A proper selective
medium is of high importance for optimizing the laboratory diagnosis of S. anginosus infections. However,conventional microbiological practices have overlooked the clinical significance of S. anginosus, because they
are frequently underrepresented on solid media.

Additionally, S. anginosus colonies are phenotypicallyindistinguishable from those of other streptococci, which traditionally have been considered to have low or no37

Fukuda et al.

Asian Pac J Dent 2012; 12: 37-44.

pathogenic potential.16,17 To overcome issues concerning the proper identification of these pathogens, several
studies have been conducted to produce a selective medium.18-20
It is an innovative idea to examine conventionally used culture media, for example, Mitis Salivarius (MS) or
Tryptic Soy (TS) agar, supplemented with antibiotics of different target spectra.21-23 Supplementing media with
antibiotics in unit volume ratios has helped to produce selective culture media to isolate certain pathogens
specifically.19,23 Bacitracin has been widely used in clinical medicine and has also been used to supplement
culture media in research laboratories,21,22 since it was first introduced in 1945.24 Bacitracin (C66H103N17O16S) is
also synthesized from cultures of Bacillus licheniformis.25 There is evidence that S. anginosus is more resistantto this antibiotic than other oral bacteria (oral streptococci other than mutans streptococci), which facilitates theselection of antibiotics to be used in this study.22
However, a culture medium that is strictly selective for S. anginosus has not yet been developed.23 Therefore,
the aim of this study was to develop a selective culture medium modified for the isolation of S. anginosus by
supplementing TS medium with antibacterial agents at optimal concentrations.

Materials and Methods
Bacterial strains used
The bacterial strains used in this study are shown in Table 1 and include streptococci from the anginosus,
mutans streptococci, mitis, and salivarius groups, as well as a staphylococcus (Staph. aureus Cowan 1). Amongthe 24 strains of oral bacteria, three clinical S. anginosus isolates were included. The laboratory strains havebeen preserved in the bacteriological facilities of our department, and the clinical isolates were kindly provided
by the Department of Oral Microbiology, Tsurumi University School of Dental Medicine.
Plate preparation
Agar plates were prepared using Tryptic Soy (TS; Becton, Dickinson and Company, Sparks, MD, USA) andagar (1.5% agarose; Wako Pure Chemical, Tokyo, Japan) and Mitis-Salivarius (MS) dissolved in distilled water
as instructed by the manufacturer. The media were autoclaved and then hardened in 10-mm diameter sterile
Petri dishes (Sansei Medical Co. Ltd., Kyoto, Japan).
Table 1. List of 24 strains of oral bacteria used in this study
Streptococcus anginosus ATCC 33397
Streptococcus anginosus TU-C20
Streptococcus anginosus TU-C21
Streptococcus anginosus TU-C25
Streptococcus intermedius ATCC 27335
Streptococcus constellatus ATCC 27823
Streptococcus mutans ATCC 25175
Streptococcus sobrinus B13N
Streptococcus sobrinus ATCC 33478
Streptococcus sobrinus 6715
Streptococcus rattus ATCC 19645
Streptococcus ferus ATCC 33477

Streptococcus cricetus ATCC 19642
Streptococcus downei ATCC 33748
Streptococcus macacae ATCC 35911
Streptococcus mitis ATCC 6249
Streptococcus salivarius ATCC 9759
Streptococcus oralis ATCC 35037
Streptococcus gordonii ATCC 10558
Streptococcus sanguinis ATCC 10556
Streptococcus pyogenes GTC 262
Streptococcus agalactiae GTC 1234
Streptococcus pneumoniae ATCC 33400
Staphylococcus aureus Cowan 1

Supplementation with antibiotics
Initially, sulphamethazine (s) (C10H10N4O2S) and aztreonam (a) (C13H17N5O8S2) (Sigma-Aldrich, St. Louis,
MO, USA) were added to TS and MS agar media to prepare TS-sa and MS-sa culture plates as mentioned above.

38

Fukuda et al.

Asian Pac J Dent 2012; 12: 37-44.

Later, three antibiotics, including the aforementioned two and bacitracin (b), were added to TS agar culture
medium to prepare TS-sab agar culture medium. Sulphamethazine and aztreonam were used at 1.0 mg/mL and
0.2 mg/mL concentrations, respectively, throughout the study.
Minimal inhibitory concentrations test
To determine the optimal bacitracin concentration for the selective growth of S. anginosus on TS agar plates, aminimal inhibitory concentration (MIC) test was performed by using 96-well plate using TS broth with seriallydiluted bacitracin. Each bacterium was cultured in TS broth for 16 hours, and 10 µL of bacterial cell suspension
was plated in each well. The plates were incubated under anaerobic (80% N2, 10% CO2 and 10% H2) condition
at 37ºC for 48 hours. Then, the turbidity of each well at 540 nm was determined by microplate reader (Bio-Rad
Laboratories, Inc., CA, USA). The spot (well), in which bacterial growth was not observed, was considered as
MIC. Subsequently, TS-sab agar plates were prepared using bacitracin concentrations of 3.2, 2.5, 2.4, 2.3, 2.2,
2.1 2.0 and 0 U/mL separately to determine a practically proper concentration of bacitracin. Five µL of eachbacterial cell suspension was inoculated directly on the agar plate and was incubated under anaerobic condition
at 37ºC for 48 hours. After this incubation, the culture plates were examined using a desktop microscope andcomparative visual scale. Visible colonies were considered indicative of bacterial growth.
Growth assessment by colony counting
Each bacterium was cultured in TS broth for 16 hours, and the bacterial cell suspension was diluted properlywith phosphate-buffered saline (PBS) and plated on TS and TS-sab agar with a spiral plating instrument (Eddy
Jet, IUL, Barcelona, Spain). All plates were incubated for 48 hours under anaerobic conditions at 37°C. After
48 hours, colony-forming units (CFUs) were enumerated with the aid of a microscope.

Results
The results of the primary assessment test are shown in Table 2.
Table 2. Primary growth assessment of the oral bacteria on TS agar, TS-sa agar, MS agar, and MS-sa agar
Bacterial strains
S. anginosus ATCC 33397
S. anginosus TU-C20
S. intermedius ATCC 27335
S. constellatus ATCC 27823
S. mutans ATCC 25175
S. sobrinus ATCC 33478
S. rattus ATCC 19645
S. ferus ATCC 33477
S. cricetus ATCC 19642
S. downei ATCC 33748
S. macacae ATCC 35911
S. mitis ATCC 6249
S. salivarius ATCC 9759
S. oralis ATCC 35037
S. gordonii ATCC 10558
S. sanguinis ATCC 10556
S. pyogenes GTC 262
S. agalactiae GTC 1234
S. pueumoniae ATCC 33400
Staph. aureus Cowan 1

TS
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

TS-sa
+
+
+
+
+
+
+
+
-

39

MS
MS-sa
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+: colony detected, -: no colony detectedFukuda et al.

Asian Pac J Dent 2012; 12: 37-44.

Table 3. Minimal inhibition concentration (MIC) of bacitracin to resist the growth of various oral bacteria
Bacterial strains
S. anginosus ATCC 33397
S. anginosus TU-C20
S. anginosus TU-C21
S. anginosus TU-C25
S. constellatus ATCC 27823
S. intermedius ATCC 27335
S. mutans ATCC 25175
S. sobrinus B13N
S. sobrinus ATCC 33478
S. sobrinus 6715
S. rattus ATCC 19645
S. ferus ATCC 33477

Bacitracin (U/mL)
6.4
6.4
6.4
0.8
3.2
0.8
0.4
0.8
3.2
3.2
3.2
<0.1

Bacterial strains
S. cricetus ATCC 19642
S. downei ATCC 33748
S. macacae ATCC 35911
S. mitis ATCC 6249
S. salivarius ATCC 9759
S. oralis ATCC 35037
S. gordonii ATCC 10558
S. sanguinis ATCC 10556
S. pyogenes GTC 262
S. agalactiae GTC 1234
S. pueumoniae ATCC 33400
Staph. aureus Cowan 1

Bacitracin (U/mL)
3.2
0.8
0.2
0.2
<0.1
6.4
0.4
0.2
<0.1
0.2
<0.1
12.8

Table 4. Determination of bacitracin concentration for selective growth of S. anginosus group on TS-sab agar
Bacitracin (U/mL)
S. anginosus ATCC 33397
S. anginosus TU-C20
S. anginosus TU-C21
S. anginosus TU-C25
S. intermedius ATCC 27335
S. constellatus ATCC 27823

6.4
+
-

3.2
+
-

2.5
+
-

2.4
+
-

2.3
+
-

2.2
+
+
+
+
-

2.1
+
+
+
+/+
-

2.0 1.8 1.0 0
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+/- +
+
+
+
+
+
+/- indicate ‘no growth’ once out of 3

Table 5. Growth characteristics of oral bacteria cultivated on TS-sab agar
Bacterial strains
S. anginosus ATCC 33397
S. anginosus TU-C20
S. anginosus TU-C21
S. anginosus TU-C25
S. intermedius ATCC 27335
S. constellatus ATCC 27823
S. mutans ATCC 25175
S. sobrinus B13N
S. sobrinus ATCC 33478
S. sobrinus 6715
S. rattus ATCC 19645
S. ferus ATCC 33477
S. cricetus ATCC 19642
S. downei ATCC 33748
S. macacae ATCC 35911
S. mitis ATCC 6249
S. salivarius ATCC 9759
S. oralis ATCC 35037
S. gordonii ATCC 10558
S. sanguinis ATCC 10556
S. pyogenes GTC 262
S. agalactiae GTC 1234
S. pneumoniae ATCC 33400
Staph. aureus Cowan 1

CFU
TS agar
2.20×108
2.48×107
1.83×107
2.27×107
2.24×107
7.30×107
1.65×108
3.74×106
3.74×106
4.39×106
2.17×108
1.48×108
5.87×106
1.57×108
1.80×108
1.39×108
1.19×108
5.08×107
2.19×108
2.16×107
9.26×107
3.52×106
4.23×106
1.67×108TS-sab agar
2.02×108
2.33×107
1.02×107
6.50×104
2.80×104
ND
ND
ND
1.22×103
4.07×102
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND

Detection rate (%)
91.6
94.0
55.7
0.3
0.1
ND
ND
ND
0.03
0.01
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND: not detected

The growth of colonies on TS and TS-sa agar media varied; most of the oral bacteria, except S. anginosus anda few other strains, did not show growth potential on TS-sa agar medium. Expectedly, all strains of S. anginosus

40

Fukuda et al.

Asian Pac J Dent 2012; 12: 37-44.

showed growth potential on all types of media when cultured using the same microbiological methods. Similar
results were observed with MS media (MS and MS-sa agar). However, TS-sa displayed slightly more resistanceto bacterial growth over MS-sa in terms of the number of bacterial strains able to grow on each medium. As aresult, TS-sa agar was chosen for further modification and analysis.
Table 3 shows the results of MIC of bacitracin against various bacteria. Ten strains of bacteria out of 24
strains were resistant to bacitracin showing MIC of bacitracin at more than 3.2 U/mL. The MIC against threestrains of S. anginosus was 6.4 U/mL. The MIC of bacitracin was found to be 2.0 U/mL, which was theconcentration used to make TS-sab agar, a medium potentially more selective for S. anginosus (Table 4).
Colony growth did not show repeatability for S. intermedius: although S. intermedius grew in some plates, it
failed to grow in others. Upon follow-up growth characterization, we found that all S. anginosus strains showedremarkable growth with high detection rates (Table 5), especially S. anginosus ATCC 33397 (93.5%) and S.
anginosus TU-C 20 (94%). Other than S. anginosus, S. intermedius and S. sobrinus grew on this at negligible
rate (0.01-0.1%). Similar results were observed in growth assays on TS-sab agar (Fig. 1). However, as shown in
Table 6, MS-sab displayed a distinct effect - no bacterial growth could be detected.
Table 6. Comparison of bacterial growth characteristics on TS-sab agar and MS-sab agar
TS-sab agar
CFU

Bacterial strains
S. anginosus ATCC 33397
S. anginosus TU-C20
S. anginosus TU-C21
S. anginosus TU-C25
S. intermedius ATCC 27335
S. constellatus ATCC 27823
S. sobrinus ATCC 33478

TS agar
+
+
+
+
+
+
+

Detection rate (%)

TS-sab agar

2.4×106
1.8×106
2.1×106
2.0×106
2.0×106
5.8×105
1.6×106

+
+
+
+
ND
ND
ND

2.4×106
1.7×104
1.3×106
7.0×105
0
0
0

94.8
91.9
64.0
35.8
0
0
0

MS-sab agar
CFU

Bacterial strains
S. anginosus ATCC 33397
S. anginosus TU-C20
S. anginosus TU-C21
S. anginosus TU-C25
S. intermedius ATCC 27335
S. constellatus ATCC 27823
S. sobrinus ATCC 33478

MS agar
+
+
+
+
+
+
+

Detection rate (%)

MS-sab agar

2.1×106
5.0×105
1.6×106
1.1×106
1.6×106
4.4×105
4.8×105

ND
ND
ND
ND
ND
ND
ND

0
0
0
0
0
0
0

0
0
0
0
0
0
0
ND: not detected

Discussion
Selective media allow certain types of organisms to grow and inhibit the growth of other organisms. This
selectivity can be accomplished in several ways. For example, organisms that can utilize a particular sugar canbe easily selected by making that sugar the only carbon source in the medium. On the other hand, selective
inhibition of some types of microorganisms can be achieved by adding antibiotics, salts, dyes, or specificinhibitors that affect the metabolism or enzyme systems of the organisms. For example, media containing
potassium tellurite, sodium azide, or thallium acetate (at concentrations of 0.1-0.5 mg/mL) will inhibit the41

Fukuda et al.

Asian Pac J Dent 2012; 12: 37-44.

growth of Gram-negative bacteria.26 Media supplemented with penicillin (5-50 U/mL) or crystal violet (2µg/mL) will inhibit the growth of Gram-positive bacteria.27 Tellurite agar, therefore, is used to select forGram-positive organisms, and nutrient agar supplemented with penicillin can be used to select forGram-negative organisms.

1) S. cricetus ATCC 19642
2) S. ferus ATCC 33477
3) S. rattus ATCC 19645
4) S. agalactiae GTC 1234
5) S. pueumoniae ATCC 33400 6) S. oralis ATCC 35037
7) S. macacae ATCC 35911 8) S. downei ATCC 33748
9) S. sanguinis ATCC 10556
10) S. gordonii ATCC 10558
11) S. mutans ATCC 25175 12) S. mitis ATCC 6249
13) S. salivarius ATCC 9759
14) S. sobrinus ATCC 33478 15) S. sobrinus 6715
16) S. sobrinus B13N17) S. pyogenes GTC 262
18) S. pyogenes GTC 262
19) S. anginosus ATCC 33397
20) S. anginosus TU-C20
21) S. anginosus TU-C21
22) S. anginosus TU-C25
23) S. constellatus ATCC 27823
24) S. intermedius ATCC 27335 25) Staph. aureus Cowan 1
Fig. 1.

Photograph showing growth of bacteria after cultivating on TS and TS-sab.
All the strains showed active growth on TS but only few (mainly S. anginosus)
grew on TS-sab (bold and underlined).

In this study, three different antibiotics were chosen to develop a selective culture medium for S. anginosus
because this group of bacteria has been reported to be involved in multiple infectious diseases of delicate organsof the human body and, most importantly, has been found in sarcomas and oral and esophageal cancers.28,29 The
development of easy, efficient, and cost-effective methods to obtain a confirmatory diagnosis in the clinic are of
the utmost importance, even in modern-day medicine and dentistry. This is no different for the confirmatory
diagnosis of S. anginosus, for two reasons: the presence of this species of bacteria is inconsistent, and there is noestablished detection method for a confirmatory etiological diagnosis or, more specifically, there is no selective
culture medium available. Although bacitracin is well known for its antibacterial potency, the addition of thisantibiotic to conventional culture media to develop a selective culture medium for confirmatory diagnosis has
not been reported before.
The addition of bacitracin, sulphamethazine, and aztreonam produced a modified TS agar medium for S.
anginosus that showed considerable selectivity and reproducibility in this study. Bacitracin has been widelyused in MS agar medium21,22 to achieve selective growth of mutans streptococci. The addition of the other two
antibiotics is probably responsible for the change in selectivity, as reported previously.22 In contrast, it has beenreported that bacitracin can be used to distinguish Streptococcus pyogenes from other bacteria.30 To develop a
new selective culture medium for S. anginosus, three antibacterial agents (sulphamethazine, aztreonam, andbacitracin) were added to tryptic soy medium to form TS-sab in this study. At this stage, reliable selectivity was42

Fukuda et al.

Asian Pac J Dent 2012; 12: 37-44.

observed only for S. anginosus ATCC 33397 at concentrations of bacitracin from 2.0 to 2.3 U/mL. Nevertheless,bacitracin at 2.0 U/mL represented the point at which most strains of S. anginosus survived, but the growth ofother bacteria could not be detected. Therefore, remarkable progress was achieved in the development of a
selective culture medium for S. anginosus in this study. However, some clinical isolates of S. anginosus were
not detected at the same high rates as the lab strains. In addition, S. sobrinus ATCC 33478 from the mutans
streptococci group showed strong resistance to bacitracin in this study. Variations in the results might beobvious as the S. anginosus species comprises 16S rRNA ribogroups that differ in phenotypic characteristics andclinical relevance.31

Therefore, TS-sab may serve as a semi-selective culture medium for S. anginosus.

Continued studies are required to develop an appropriate selective medium for confirmatory clinical diagnosis byusing a series of antibacterial agents and peptides at various concentrations and also using various bacterial
species. In the oral cavity, antimicrobial peptides, such as defensin, histain, and cathelicidin, play a pivotal role
as a first line of defense against a succession of invading bacteria.32 The β-defensins are small, cationic,
antimicrobial peptides. The peptides hBD-1 and -3 were detected in the salivary gland and gingival, tongue, andbuccal mucosa. Such peptides should be considered to supplement TS-sab for the specific identification of S.
anginosus.
Acknowledgment
We would like to express our gratitude to Dr. Y. Nomura and Dr. A. Okada, Department of Translational Research,
Tsurumi University School of Dental Medicine, for their valuable advice. We thank to Dr A. Takao, Department of Oral
Microbiology, Tsurumi University School of Dental Medicine, for providing clinical isolates of S. anginusus. We also
acknowledge the contribution of Dr. K. Matin, Cariology Operative Dentistry, Department of Restorative Science, Graduate
School, Tokyo Medical and Dental University, in preparing the manuscript.

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Correspondence to:
Dr. Susumu Imai
Department of Translational Research, Tsurumi University School of Dental Medicine
2-1-3 Tsurumi, Tsurumi-ku, Yokohama 230-8501, Japan
Fax: +81-45-573-2473 E-mail: imai-s@tsurumi-u.ac.jp
Accepted December 22, 2012.
Copyright ©2012 by the Asian Pacific Journal of Dentistry.
Online ISSN 2185-3487, Print ISSN 2185-3479

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