Journal of Medical Microbiology (2011), 60, 17011704 DOI 10.1099/jmm.0.

030114-0

Case Report Streptococcus australis meningitis

Genevieve Hery-Arnaud,1,2 Nicolas Rouzic,3 Alexandra Doloy,4
Genevieve Le Lay,1 Michel Garre,3 Christopher Payan1,2 and Claire Poyart4
Correspondence 1
Departement de Microbiologie, Centre Hospitalier Regional Universitaire (CHRU) Brest, France
Genevieve Hery-Arnaud 2
EA 3882-IFR 148, Universite Bretagne Ouest, Brest, France
hery@univ-brest.fr
3
Service des Maladies Infectieuses, CHRU Brest, France
4
Universite Paris Descartes, Centre National de Reference des Streptocoques, Service de
Bacteriologie, Groupe Hospitalier Cochin-Saint-Vincent de Paul, AP-HP, Paris, France

Received 11 January 2011 We report a case of meningitis due to Streptococcus australis, a species of oral streptococcus.
Accepted 4 July 2011 Accurate identification was performed by various molecular techniques.

Case report
A 77-year-old man was admitted to the infectious diseases
unit of CHRU Brest with a 3-day history of fever, nausea,
vomiting and growing headache. On admission, the patient
displayed full-blown meningitis, including coiled position-
ing. Chest radiography revealed left-side infiltrate in line
with crackling sounds but without clinical signs. The
patient was rapidly transferred to the intensive care unit
because of severe sepsis, and empirical antibiotic treatment
was started combining amoxicillin [200 mg kg21 per day,
administered intravenously (i.v.)] and cefotaxime (200 mg
kg21 per day i.v.); the patient was also given dexametha-
sone (10 mg every 6 h). Three hours after first admin-
istration of antibiotics, lumbar puncture yielded purulent
cerebrospinal fluid (CSF) with pleiocytosis (64006106
white blood cells l21 with 98 % polymorphonuclear
leukocytes and 6006106 red blood cells l21), decreased
CSF glucose concentration (0.25 g l21) and increased CSF
protein concentration (6.47 g l21). No bacteria were
observed on CSF Gram staining. A set of two blood
cultures performed on admission, before receiving anti-
biotics, was positive. Direct examination by Gram staining
showed Gram-positive cocci organized into chains subse-
quently identified as Streptococcus australis strain
CNRCCH2009401. A second set of blood cultures was
performed 4 h after initial antibiotic treatment and
remained sterile. CSF solid culture remained negative, as
did the Schaedler broth inoculated with the CSF, even after
10 days of incubation. The CSF sample was tested by
broad-range 16S rRNA gene PCR combined with sequen-
cing, which yielded Streptococcus species secondly identified
as S. australis by sodA gene sequencing. On the basis of
antibiotic susceptibility testing conducted on the blood
culture isolate, cefotaxime was stopped and amoxicillin was
Abbreviation: CSF, cerebrospinal fluid.
The GenBank/EMBL/DDBJ accession number for the sodA sequence
of the blood isolate CNRCCH2009401 is HM189677.
administered i.v. for 1 week. The patients status improved
rapidly. Results from the cerebral CT scan and transtho-
racic echocardiography were normal. According to Duke
criteria, a diagnosis of infective endocarditis was elimi-
nated. Follow-up visits showed a healthy patient with no
clinical signs of relapse.
Microbiological findings
The S. australis strain grew as a-haemolytic colonies on
both horse and sheep blood agar (Oxoid) incubated for
18 h at 37 uC under aerobic and anaerobic conditions. We
observed Gram-positive, catalase-negative cocci organized
into small chains. Biochemical characteristics of the strain
obtained with the Rapid ID 32 STREP system (bioMerieux)
(profile 44012441100) corresponded to Streptococcus oralis
or Streptococcus mitis with accuracies of 56.1 % and 43.5 %,
respectively. Vitek II identification performed with a GP
card (bioMerieux) resulted in the strain being identified as S.
oralis or S. mitis as well (profile 065510364351011) with
accuracies of 93 %. Antibiotic susceptibility testing was
performed on MuellerHinton sheep blood agar (Oxoid)
using the disc diffusion method and MICs were determined
with the Etest (AES). The Antibiogram Committee of the
French Society for Microbiology criteria for Streptococcus
species were used for the interpretation of antimicro-
bial drug susceptibility (http://www.sfm-microbiologie.org).
Strain CNRCCH2009401 was susceptible to b-lactams
(MICs of benzylpenicillin, amoxicillin and cefotaxime
,0.016 mg ml21), tetracycline, erythromycin, pristinamy-
cin, clindamycin, rifampicin, fosfomycin, gentamicin (disc
concentration of 500 mg), kanamycin (disc concentration of
1000 mg), vancomycin and teicoplanin. Universal 16S rRNA
gene amplification was performed on CSF to confirm
bacterial meningitis. DNA extraction was performed with
the QIAamp DNA Mini kit (Qiagen), and PCR was
performed with the universal prokaryotic primers F8-27
(59-AGAGTTTGATCCTGGCTCAG-39) (Koort et al., 2005)

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G. Hery-Arnaud and others

and RU2 [59-TACGG(T/C)TACCTTGTTACGACTTC-39]; Streptococcus sanguinis, Streptococcus parasanguinis, S. mitis,

these primers were adapted from those used by Lu et al.
(2000) to amplify a segment of approximately 1500 bp
corresponding to part of the 16S rRNA gene. Semi-nested
PCR with the primers U1 (59-CCAGCAGCCGCGGTAAT-
ACG-39) and RU2 (Lu et al., 2000) generated a 996 bp
segment, which was sequenced. BLAST sequence analysis
yielded 98 % identity with sequences for streptococcal species
that belong to the Streptococcus mitis group, such as S. oralis,
Streptococcus infantis, Streptococcus pneumoniae and
Streptococcus pseudopneumoniae (NCBI and BIBI databases).
Accurate identification at the species level was performed by
sodA gene amplification and sequencing, as previously
described (Poyart et al., 1998). Sequence analysis yielded
97 % identity with the sequence for the S. australis type strain
CIP 107167 (GenBank accession no. DQ132987). Identical
results were obtained with the blood culture isolate.

Table 1. Phenotypic characteristics of the Streptococcus australis isolate from this study compared with those of S. australis isolates
described previously
The S. australis isolate from this study was compared to those described in previous reports (Willcox et al., 2001; Hoshino et al., 2005). +, 85 % or
more of the strains tested positive; 2, 15 % or less of the strains tested positive; v, 1684 % of the strains tested positive. All the strains were typed by
the Rapid ID 32 STREP system (bioMerieux). ND, Not determined.

Phenotypic characteristic Isolate CNRCCH2009401 Isolates from previous studies*

Willcox et al. (2001) Hoshino et al. (2005)

Arginine dihydrolase 2 + v
b-Glucosidase 2 2 2
b-Galactosidase* + + v
b-Glucuronidase 2 2 2
a-Galactosidase 2 2 2
Alkaline phosphatase + + 2
Ribose acidification 2 2 2
Mannitol acidification 2 2 2
Sorbitol acidification 2 2 2
Lactose acidification + + +
Trehalose acidification 2 2 2
Raffinose acidification 2 2 2
Acetoin production 2 2 2
Alanyl-phenylalanyl-proline-arylamidase + + +
b-GalactosidaseD 2 2 2
Pyroglutamic acid arylamidase 2 2 2
N-Acetyl-b-glucosaminidase 2 2 2
Glycyl-tryptophan arylamidase + + v
Hydrolysis of hippurate 2 2 2
Glycogen acidification 2 2 2
Pullulane acidification + + v
Maltose acidification + + +
Melibiose acidification 2 2 2
Melezitose acidification 2 2 2
Sucrose acidification + + +
L-Arabinose acidification 2 2 2
D-Arabitol acidification 2 2 2
Methyl b-D-glycopyranoside acidification 2 2 2
Tagatose acidification 2 2 v
b-Mannosidase 2 2 2
Cyclodextrin acidification 2 2 2
Urease 2 2 ND
Site of isolation (number of strains) Blood (n51) Saliva (n510)d Mouth (n51)d and blood (n51)
Clinical context Meningitis Carriage Endocarditis and carriage

*Detected with p-nitrophenyl b-D-galactopyranoside as the substrate.

DDetected with 2-naphthyl b-galactopyranoside as the substrate.
dIncluding the reference strain ATCC 700641T.

1702 Journal of Medical Microbiology 60


Discussion
S. australis, an oral streptococcal species, was first described
in 2001 from strains isolated during a microbiological
survey of saliva samples from healthy children attending
the United Hospital in Sydney, Australia (Willcox et al.,
2001). S. australis belongs to the S. mitis group, one of the
five groups of viridans streptococci described by Facklam
(2002). Standard biochemical phenotypic identification,
such as the Rapid ID 32 STREP system (bioMerieux),
showed that isolate CNRCCH2009401 resembled S. oralis
and S. mitis. Comparing isolate CNRCCH2009401 with the
S. australis isolates described by Willcox et al. (2001) and
Hoshino et al. (2005), we noted that, even though the
Rapid ID 32 STREP system (bioMerieux) was employed in
all three studies, there were differences in the phenotypic
characteristics (Table 1). Therefore, phenotypic character-
ization is of limited value during the identification of several
viridans streptococci species (Hoshino et al., 2005; Jacobs
et al., 1995). Phylogenetic analysis based on 16S rRNA gene
sequencing confirmed that isolate CNRCCH2009401
belongs to the S. mitis group. There is significant sequence
conservation of the 16S rRNA gene; thus, this molecular tool
is not appropriate for accurate identification in this group of
streptococci (Hoshino et al., 2005; Kawamura et al., 1999;
Matta et al., 2009; Poyart et al., 1998). The sodA gene has
been identified as a valuable target housekeeping gene for
sequence-based identification, as the results obtained with
this gene are consistent with conclusive identification
(Poyart et al., 1998). In this study, sodA gene sequence
typing identified isolate CNRCCH2009401 as S. australis
with 97 % identity with the type strain, CIP 107167.
Remarkably, sodA gene sequences of S. pneumoniae, S. mitis
and S. oralis shared between 93 % and 94 % identity with the
sequence of the S. australis type strain. Therefore, unam-
biguous sodA sequences are essential to conduct an accurate
molecular diagnosis of S. australis. Finally, diagnosis of S.
australis must be the result of a combination of both
phenotypic and molecular robust arguments.
Members of the S. mitis group are some of the most
frequently isolated non-b-haemolytic streptococci in bac-
teraemic patients (Hoshino et al., 2005). These species are
usually responsible for subacute endocarditis (Hoshino
et al., 2005), but are rarely responsible for community-
acquired meningitis (Cabellos et al., 1999). The few cases of
meningitis reported to be due to these streptococcal species
were associated with either an iatrogenic origin (Idigoras
et al., 2001; Legrand et al., 2007; Yaniv & Potasman, 2000),
such as dental manipulation, invasive spine investigation,
spinal anaesthesia and gastrointestinal endoscopy, or an
underlying disease (Hoshino et al., 2005; Idigoras et al.,
2001; Jacobs et al., 1995; Yaniv & Potasman, 2000), such as
neutropenia and malignancy. For most of the cases of
community-acquired acute meningitis caused by S. mitis
group streptococci, co-morbidities have been suggested
(Kutlu et al., 2008; Mller et al., 1999). In our case, the
patient presented no surgical or traumatic history; the
predisposing factors were old age and an untreated
http://jmm.sgmjournals.org
Streptococcus australis meningitis
monoclonal gammopathy of undetermined significance.
Considering these underlying conditions, it appears that
our patient presented initial pneumonia complicated with
secondary meningitis by haematogen diffusion. Negative-
CSF culture can be explained by preliminary antibiother-
apy and low inoculum density.
In conclusion, we report to the best of our knowledge the
first documented case of an invasive S. australis infection
resulting in meningitis. The molecular diagnostic approach
in this study should be useful for assessing the occurrence
of S. australis in other human infections, which, in the past,
have primarily been thought to be due to S. mitis or S.
oralis isolates. Nevertheless, even in the age of molecular
tools, it remains extremely difficult to make an accurate
identification of viridans streptococci.
Acknowledgements
We are grateful to Christine Le Guen and to Annick Billoet for their
excellent technical assistance. We thank Francoise Geffroy for her
insightful contribution.
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