JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1998, p.

866871
0095-1137/98/$04.0010
Copyright 1998, American Society for Microbiology

Vol. 36, No. 4

Molecular Identification of Gemella Species from

Three Patients with Endocarditis
BERNARD LA SCOLA

AND

DIDIER RAOULT*

Unite des Rickettsies, CNRS UPRESA 6020, Faculte de Medecine, Universite de la

Mediterrannee, 13385 Marseille Cedex 05, France
Received 31 July 1997/Returned for modification 10 October 1997/Accepted 30 December 1997

Gemella morbillorum and Gemella haemolysans are grampositive coccal commensal organisms of the mucous membranes of humans and other warm-blooded animals. However,
as opportunistic pathogens, gemellae are able to cause severe localized and generalized infections.
The cases of Gemella infection reported to date have been
predominantly endovascular infections (1, 6, 812, 15, 22, 25,
28, 29, 31, 3335, 39, 40, 43, 45). Among these cases of Gemella
infection, most are endocarditis, usually associated with previous valvular damage and/or poor dental state. Central nervous
system and skeletal infections have also been described (4, 13,
23, 36, 38, 48). In a study of 52 cases of streptococcal endocarditis, gemellae represented 6% of the viridans group streptococci and 5% of all isolates (17). In our hospital center,
gemellae were the cause of 6% of the cases of endocarditis due
to nonstaphylococcal gram-positive cocci, were the cause of
13.3% of the cases of endocarditis caused by viridans group
streptococci, and represented 2.9% of all bacterial isolates
causing 70 cases of endocarditis diagnosed during a 3-year
period (unpublished data).
These bacteria are easily decolorized during Gram staining
and sometimes appear as elongated cells, explaining why they
were first described as Neisseria (46). They may also be more
involved in clinical disease than is presently recognized, because they can be incorrectly identified as viridans group streptococci or left unidentified (42).
In this paper we report on two patients with G. morbillorum
endocarditis and one patient with G. haemolysans endocarditis.
The identification of one G. morbillorum strain was achieved
with the help of partial 16S rRNA gene sequencing, since the
bacterial isolate appeared to be gram negative. Partial 16S
rRNA gene sequencing was also used as a reference technique
to confirm the identities of a further two strains of Gemella.
The results obtained by electron microscopy and analysis of

cell wall fatty acids are reported, and the previous cases of
endocarditis caused by G. haemolysans and G. morbillorum are
reviewed.
CASE REPORTS
Patient 1. A 63-year-old man who was a heavy smoker with
chronic obstructive bronchitis and a very poor dental state was
admitted to hospital complaining of intermittent fever, loss of
weight, and anorexia over a 1-month period. He had no history
of rheumatic disease, although a moderate heart murmur had
been discovered 1 year previously, and at that time an echocardiogram had yielded moderate mitral valve regurgitation.
On initial examination, the patient had an increased heart
murmur, a temperature of 38.5C, and a pulse of 100 beats/
min. An ejection systolic murmur was heard at the apex of the
heart. Laboratory investigations showed a hemoglobin concentration of 100 g/liter, an erythrocyte sedimentation rate of 97
mm/h, and a leukocyte count of 8.53 3 109/liter (77% neutrophils). A transesophageal echocardiogram demonstrated the
presence of vegetation on the mitral valve with moderate mitral valve regurgitation. Six sets of blood samples for cultures
were taken on admission and the following day, and the blood
samples were inoculated into BACTEC aerobic (NR 6-A*)
and anaerobic (NR-7A*) bottles in a BACTEC NR-860 automated instrument (Becton Dickinson Diagnostic Instrument
Systems, Sparks, Md.). All yielded slowly growing, gram-positive cocci, subsequently identified as G. haemolysans. A kidney
echography, carried out for microscopic hematuria, yielded a
lesion inside of the left kidney which was compatible with an
abscess. The patients treatment began the day after his admission, in which treatment with amoxicillin (4 g intravenously at
6-h intervals) and amikacin (5 mg/kg of body weight intravenously at 8-h intervals) was begun. His condition improved
rapidly, and after 2 weeks of this regimen, he underwent cardiac surgery in order to remove the motile vegetation. One
week after surgery antibiotic therapy was discontinued. After 2
years of follow-up he remains well.
Patient 2. A 74-year-old man with a history of Potts disease
in infancy, chronic alcoholism, and a poor dental state was

* Corresponding author. Mailing address: Unite des Rickettsies,

CNRS UPRESA 6020, Faculte de Medecine, Universite
de la Mediterrannee, 27 Blvd. Jean Moulin, 13385 Marseille Cedex 05, France.
Phone: (33).4.91.38.55.17. Fax: (33).4.91.83.03.90. E-mail: Didier
.Raoult@medecine.univ-mrs.fr.
866

Downloaded from http://jcm.asm.org/ on June 9, 2015 by guest

Gemella morbillorum and Gemella haemolysans are opportunistic pathogens which cause endocarditis and
other severe infections. We report on three patients with endocarditis, one with endocarditis caused by G.
haemolysans and two with endocarditis caused by G. morbillorum. The paucity of reports concerning these
bacteria is probably related to the difficulties associated with their identification. For example, one of the
strains reported in this study was originally sent to our laboratory with a preliminary characterization as a
short gram-negative coccobacillus, highlighting the specific problem associated with Gram staining of these
bacteria. The usefulness of 16S rRNA gene amplification, partial sequencing, and comparison of the nucleotide
sequence to those in databases when standard phenotypic identification schemes are not helpful is emphasized.
We also suggest that the use of simple tests, such as testing susceptibility to vancomycin for gram-negative
bacteria and colistin for gram-positive bacteria, could prevent misinterpretation of Gram staining in gramvariable bacteria such as Gemella spp.

MOLECULAR IDENTIFICATION OF GEMELLA SPECIES

VOL. 36, 1998

MATERIALS AND METHODS

Phenotypic identification. Presumptive identification of the Gemella organisms was achieved through assessment of colonial morphology, hemolysis on
Columbia agar with 5% sheep blood (BioMerieux, Marcy lEtoile, France),
microscopic appearance after Gram staining, and the results of biochemical tests
(with the API 20 Strep and the API 20A systems according to the manufacturers
instructions). Growth was also attempted in broth containing 6.5% NaCl. Susceptibility to vancomycin and colistin was assessed with 30-mg vancomycin and
50-mg colistin disks (Sanofi Diagnostic Pasteur, Marnes la Coquette, France) and
Mueller-Hinton broth with 5% sheep blood agar (BioMerieux) by the conventional disk diffusion test method (2). The strain was considered to be susceptible
to vancomycin and to colistin when inhibition zone diameters of $12 and $15
mm, respectively, were observed.
Cellular fatty acids. Colonies of the three isolates and of the type strains G.
haemolysans ATCC 10379 and G. morbillorum ATCC 27824 were grown on
Trypticase soy agar with 5% sheep blood (Becton Dickinson) at 38C for 48 h.
They were then saponified, and cell wall fatty acids were extracted and analyzed
by gas chromatography as reported previously (37).
Processing for electron microscopy. Bacterial cells were harvested from colonies grown on Columbia agar and for fixation were suspended in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) containing 0.1 M sucrose. The fixed
cells were washed overnight with the same buffer and were then fixed for 1 h at
room temperature with 1% osmium tetroxide in 0.1 M cacodylate buffer. Dehydration was performed by washing the cells in gradually increased concentrations
(25 to 100%) of ethanol. The cells were then embedded in Epon 812. Thin
sections were cut from embedded blocks with an LKB Ultratome III microtome
and were poststained with a saturated solution of methanol-uranyl acetate and
lead citrate in water before examination on a Jeol JEM 1200 EX electron
microscope.

TABLE 1. Summary of primers used for PCR amplification and

sequencing in this study
Primer
purpose and
primer

PCR
fD1
rP2
Sequencinga
fD1
rP2
800f
800r
1050f
1050r
a

Sequences (59 to 39)

-AGAGTTTGATCCTGGCTCAG-ACGGCTACCTTGTTACGACTT-

-AGAGTTTGATCCTGGCTCAG-ACGGCTACCTTGTTACGACTT-ATTAGATACCCTGGTAG-CTACCAGGGTATCTAAT-TGTCGTCAGCTCGTG-CACGAGCTGACGACA-

Primer
specificity or
hybridization
temp (C)

Eubacterial 16S
rRNA gene
Eubacterial 16S
rRNA gene
55
55
50
50
43
43

Primers 59 labelled with fluorescein.

PCR amplification and sequencing of the PCR product. DNA extracts, suitable for use as templates in PCRs, were made from 200 ml of a bacterial
suspension by using a QIA ampBlood kit (Qiagen, Hilden, Germany) according
to the manufacturers instructions. All primer sets used in this study are listed in
Table 1. DNA extracts were amplified by a PCR incorporating universal primers
fD1 and rP2 (49) (Eurogentec, Seraing, Belgium). PCR amplifications were
performed in 100-ml volumes incorporating 10 ml of extracted DNA, 10 ml of 103
reaction buffer, 100 mM (each) deoxynucleoside triphosphate, 0.2 mM (each)
primer, 2 U of Taq polymerase (Perkin-Elmer Cetus, Norwalk, Conn.), and
sterile, distilled water. Amplifications were carried out in a Perkin-Elmer 9600
thermal cycler for 35 cycles, with each cycle consisting of denaturation at 95C for
30 s, primer annealing at 55C for 30 s, and extension at 72C for 60 s. The
success of the amplification was determined by ethidium bromide staining following the resolution of products by 1% agarose gel electrophoresis. Each experiment included sterile water (no DNA) as a negative control and Escherichia
coli DNA as a positive control. The products of 16S rRNA gene amplification
were purified for sequencing by using Microspin S-400 HR columns (Pharmacia
Biotech, Uppsala, Sweden) according to the manufacturers instructions. Sequencing reactions were prepared by using the Amplicycle sequencing kit (Perkin-Elmer), according to the manufacturers instructions, and one of six 59
fluorescein-labelled primers was incorporated into the reaction mixture (Table
1). The thermal cycle used in each sequencing reaction was dependent on the
base sequence of the primer (Table 1). When the primer annealing temperature
was $50C, an initial denaturation step at 95C for 1 min was followed by 25
cycles, with each cycle comprising denaturation at 95C for 30 s, annealing for
30 s, and extension at 72C for 1 min. Amplification was completed by an
extension step of 5 min at 72C to allow complete extension of the amplified
products. When the primer annealing temperature was ,50C, an initial denaturation step at 95C for 1 min was followed by 30 cycles, with each cycle
comprising denaturation at 95C for 30 s, annealing for 30 s, and extension at
60C for 2 min. Ten supplementary cycles of denaturation at 95C for 10 s and
extension at 60C for 90 s were performed in order to increase the amount of
polymerization. Sequencing reactions were carried out on a Perkin-Elmer 9600
thermal cycler, and reaction products were resolved by electrophoresis on a 6%
polyacrylamide gel incorporated into an ALF Automatic Sequencer (Pharmacia
Biotech).
Sequence analysis. Partial 16S rRNA gene sequences derived from each reaction mixture with the ALF manager were combined in a complete sequence by
using PC Gene software (IntelliGenetics Inc.). The complete sequence was then
compared with all bacterial sequences available in the GenBank database, by
using the multisequence comparison program FASTA (part of the BISANCE
software package) (14).

RESULTS
Bacterium from patient 1. The bacterium isolated from patient 1 was thought to be a strain of G. haemolysans on the
basis of conventional phenotypic identification. Growth was
easily achieved on Columbia agar with incubation at 37C
under 5% CO2, but only poor growth was obtained under
anaerobic conditions. Colonies were small and weakly betahemolytic after 5 days of incubation. Microscopic examination
after Gram staining yielded gram-positive cocci which became

Downloaded from http://jcm.asm.org/ on June 9, 2015 by guest

admitted to hospital complaining of intermittent fever, sweating, loss of weight, and basithoracic pain over a 3-month period. He had neither a history of rheumatic disease nor a
previous heart murmur. On initial examination, the patient had
a diastolic heart murmur, a temperature of 38C, and a pulse of
100 beats/min. Laboratory investigations showed a hemoglobin
concentration of 95 g/liter, an erythrocyte sedimentation rate
of 63 mm/h, and a leukocyte count of 12.8 3 109/liter (87%
neutrophils). A transesophageal echocardiogram demonstrated aortic valve incompetence but failed to show any vegetation. Six sets of blood samples for culture were taken on
admission and the following day, and the blood samples were
inoculated into BACTEC aerobic (NR 6-A*) and anaerobic
(NR-7A*) bottles in a BACTEC NR-860 automated instrument. All yielded slowly growing, gram-variable cocci and coccobacilli subsequently identified as G. morbillorum. The patients treatment began the day after his admission and
comprised amoxicillin (4 g intravenously at 6-h intervals) and
gentamicin (1 mg/kg intravenously at 8-h intervals). Although
his condition improved rapidly, it was necessary to transfer him
to a cardi-thoracic unit because he was also suffering from
significant aortic valve regurgitation and a dilated left ventricle.
The aortic valve was successfully replaced with a prosthetic
device, and the patient made an uneventful postoperative recovery. Gentamicin was discontinued 1 week later, and amoxicillin was discontinued 3 weeks later. After 1 year of follow-up
he remains well.
Patient 3. A small, fastidious, gram-negative rod which had
been isolated from the blood of a male patient with infectious
endocarditis by using BACTEC aerobic (NR 6-A*) and anaerobic (NR-7A*) bottles was sent to our laboratory for identification, because it was thought that it could be a Bartonella sp.
The patient was a farmer and suffered from a bicuspid aortic
valve. Clinical symptoms included intermittent fever and a
weight loss of 12 kg over a period of several months. Attempts
to amplify citrate synthase and 16S rRNA genes with specific
primers for Bartonella (27) were unsuccessful. Standard phenotypic characterization methods for gram-negative rods also
failed to provide an identity. The bacterium was identified as
G. morbillorum by 16 rRNA gene sequencing.

867

868

J. CLIN. MICROBIOL.

LA SCOLA AND RAOULT

TABLE 2. Results of phenotypic characterization of strains isolated

in this study
Feature

Voges-Proskauer
Alkaline phosphatase
Leucine aminopeptidase
Pyrrolidone arylamidase
Esculin hydrolysis
Acid from glucose
Acid from sucrose
Acid from maltose
Acid from mannitol
Acid from mannose
Acid from trehalose
Growth in 6.5% NaCl
Vancomycin susceptibility

Patient 1,
Patient 2,
Patient 3,
G. haemolysans G. morbillorum G. morbillorum

1
1
1
1
2
1
1
1
1
1
2
2
1

2
2
1
2
2
1
1
1
2
1
1
2
1

2
2
1 (weak)
2
2
1
1
1
1
1
2
2
1

G. morbillorum was originally proposed as Diplococcus rubeolae (47), and a second member of the genus named Diplococcus morbillorum was added soon after (41). However, on
reappraisal the two Diplococcus species were considered to be
identical and were unified under the name D. morbillorum.
Although D. morbillorum could be grown under aerobic conditions, it was primarily anaerobic, and on this basis it was
transferred to the genus Peptostreptococcus (44), only to be
reclassified into the genus Streptococcus (26). G. haemolysans
was first described in 1938 as Neisseria haemolysans (46) (demonstrating its indeterminate Gram staining characteristic), but
the species was reclassified into a new genus, Gemella (7),
following demonstration of biochemical differences with other
Neisseria species. The nucleotide sequence of the 16S rRNA
gene of Streptococcus morbillorum was found to closely resemble that of G. haemolysans (32), and on the basis of DNA-DNA
hybridization results and G1C content analysis, it was proposed that S. morbillorum be transferred to the genus Gemella
as G. morbillorum comb. nov. (30). The phylogenetic relationships of Gemella spp. to other gram-positive bacteria are presented in Fig. 1.
G. morbillorum and G. haemolysans are uncommon causes
of infectious endocarditis; a review of the literature reveals
only 10 cases caused by G. morbillorum (1, 6, 10, 12, 29, 33, 35,
40, 45) and 12 cases caused by G. haemolysans (8, 9, 11, 15, 22,
25, 28, 31, 34, 39, 43). Two additional cases of G. morbillorum
endocarditis have been recorded among 52 apparent failures of
endocarditis prophylaxis (17), and 8 cases have been recorded
among 364 cases of streptococcal endocarditis (19). The patients described in Table 3 and Table 4 demonstrate that poor
dental state and/or dentistry are predisposing factors, as in
patients with endocarditis caused by viridans group streptococci. Previous valvular damage is also a common occurrence.
In most cases, the infections were successfully treated with
antibiotic therapy, usually benzylpenicillin or amoxicillin associated with gentamicin.
Gemella spp. possess a typical gram-positive cell wall structure, as confirmed by electron microscopy in this study. However during Gram staining, cells are easily decolorized and may
therefore may appear to be gram variable and even gram
negative. It is likely that Gram staining abnormality and morphological polymorphism are responsible for the misidentification of Gemella spp. and, thus, perhaps for the fact that so
few cases of Gemella infection are reported. Rapid phenotypic
identification systems are unable to identify accurately all
strains of these species (3, 20), even though manufacturers
have significantly improved their databases over recent years
(e.g., the Rapid ID 32 Strep database [21]).
Nevertheless, for two cases of infection described in this
report, identification of G. morbillorum or G. haemolysans as
the causative agents was achieved by standard phenotypic identification schemes. Identification of the organism causing infection in patient 3 was difficult, however, even though a large
number of biochemical tests were performed. After its identification had been achieved by the PCR-based approach with
the 16S rRNA gene and the bacterium had been recognized as
being gram positive, reassessment of phenotypic characteristics
revealed a correct identity. The same problem had already
occurred with a short, gram-negative bacillus, isolated from
a patient with infectious arthritis, which had been sent to our
laboratory for identification; analysis of its 16S rRNA gene also
revealed that this isolate was G. morbillorum (unpublished
data).
In our laboratory, we now routinely use cell wall fatty acid

Downloaded from http://jcm.asm.org/ on June 9, 2015 by guest

gram-variable after subculture. Cocci were arranged in pairs,

short chains, or clusters and were of various sizes. The results
of biochemical reactions and enzyme analyses are summarized
in Table 2. Sequence data for 1,022 bp of the 16S rRNA gene
were obtained. After removing ambiguities, the sequence demonstrated 99.9% similarity to the 16S rRNA gene sequence of
G. haemolysans (GenBank accession no. L14326) (50).
Bacterium from patient 2. The bacterium isolated from patient 2 was thought to be a strain of G. morbillorum on the basis
of conventional phenotypic identification. Growth was easily
obtained on Columbia agar with incubation at 37C under 5%
CO2 and under anaerobic conditions. Colonies were weakly
alpha-hemolytic only when they were incubated under 5%
CO2. Microscopic examination after Gram staining yielded
gram-variable cocci and coccobacilli arranged in pairs, short
chains, or clusters and of various sizes. The results of biochemical reactions and enzyme analyses are summarized in Table 2.
Sequence data for 948 bp of the 16S rRNA gene were obtained. After removing ambiguities, the sequence demonstrated 99.8% similarity to the 16S rRNA gene sequence of G.
morbillorum (GenBank accession no. L14327).
Bacterium from patient 3. Conventional phenotypic characterization of the gram-negative coccobacilli yielded no definite
identification. Visible growth was obtained after 3 or 4 days on
Columbia agar with incubation at 37C under 5% CO2. The
colonies were small and nonhemolytic. Sequence data for 949
bp of the 16S rRNA gene were obtained. After removing
ambiguities, the sequence demonstrated 99.68% similarity to
the 16S rRNA gene sequence of G. morbillorum (GenBank
accession no. L14327). Conventional biochemical identification was reassessed. The results are summarized in Table 2 and
are compatible with the identification of the bacterium as G.
morbillorum with the exception of the results obtained by
Gram staining. Subsequently, a more complete 16S rRNA
gene sequence was determined. This 1,474-bp sequence demonstrated 99.73% similarity with the 16S rRNA gene sequence
of G. morbillorum.
Cellular fatty acids. The major fatty acids in cells were
identified as C16 and C18, but definite identification was not
achieved by this technique.
Electron microscopy. All bacteria studied presented with a
typical gram-positive membrane organization. Some cells appeared as short bacilli, usually when they were in the process of
dividing.

DISCUSSION

MOLECULAR IDENTIFICATION OF GEMELLA SPECIES

VOL. 36, 1998

869

analysis and/or 16S rRNA gene analysis when a bacterium is

not easily identified by phenotypic schemes. About 0.05 to
0.1% of all isolates fall into this category. Amplification with
universal primers, partial sequencing (about 900 bp) with con-

served primers, and sequence comparison take less than 3 days.

If the partial 16S rRNA gene sequence obtained shares more
than 99% similarity with a specific 16S rRNA gene sequence in
the database, the bacterium may be considered to be identi-

TABLE 3. Summary of patients with reported cases of G. morbillorum endocarditis and features of our two patients with G.
morbillorum endocarditisa
Patient no./
age (yr)/sexa

Underlying conditions or source of infection

Infected
valve

1/60/m
2/38/m

Dental manipulation
Anal subcutaneous sphincterotomy,
sigmoidoscopy

3/39/m

Dental manipulation, bicuspid aortic valve Ao

4/42/m

Mi regurgitation

Mi

5/19/m

Intravenous drug abuser

Tri

6/48/m

Poor dentition, systolic heart murmur,

Mi
end-stage renal disease
Dental manipulation
Mi, Ao Benzylpenicillin
Hypertrophic obstructive cardiomyopathy, Na
Benzylpenicillin,
dental manipulation
gentamicin,
erythromycin,
rifampin
Left colonic resection for rectal neoplasia Tri
Cefuroxime,
amikacin,
benzylpenicillin
Rheumatic fever in infancy
Mi
Benzylpenicillin,
gentamicin,
teicoplanin,
rifampin,
erythromycin
Poor dentition, chronic ethylism
Aob
Amoxicillin,
gentamicin
Bicuspid aortic valve
Ao
NA

7/64/m
8/29/f

9/74/m
10/75/m

11/74/m
12/NA/m
a
b

NA
Mi

Therapy

Penicillin, rifampin
Benzylpenicillin,
gentamicin,
erythromycin
Benzylpenicillin,
gentamicin
Benzylpenicillin,
streptomycin
Flucloxacillin,
benzylpenicillin,
gentamicin
Vancomycin

Valve
replacement

Outcome

Reference,
year

No
No

Cure
Cure

12, 1984
35, 1989

Yes

Cure

35, 1989

No

Cerebral mycotic aneurysm, cure 10, 1990

No

Cure (?; reduction in the size of

vegetation)

6, 1992

No

Wrist arthritis, cure

40, 1993

Yes
No

Acute appendicitis, cure

Cure

45, 1994
29, 1994

No

Pulmonary embolism, cure

1, 1995

No

Allergic reaction to
antimicrobial agents, cure

33, 1995

Yes

Cure

Patient 2

NA

NA

Patient 3

Abbreviations: Ao, aortic valve; Mi, mitral valve; Tri, tricuspid valve; NA, not available; m, male; f, female.
No evidence of vegetation on echocardiogram.

Downloaded from http://jcm.asm.org/ on June 9, 2015 by guest

FIG. 1. Phylogenetic tree based on 16S rRNA gene sequences available in the GenBank database obtained by the neighbor-joining method. The phylogenetic
relationships of G. haemolysans and G. morbillorum with selected gram-positive bacteria with low G1C contents are shown. The bar represents a 1% difference in
nucleotide sequence.

870

LA SCOLA AND RAOULT

J. CLIN. MICROBIOL.

TABLE 4. Summary of reported cases of G. haemolysans endocarditis and features of our patient with G. haemolysans endocarditisa
Case/age
(yr)/sex

Underlying conditions or
source of infection

Infected
valve

1/62/m

Dental manipulation

Mi

2/48/m

Poor dentition, respiratory viral

syndrome
Mi insufficiency, dental manipulation
Aortic insufficiency
Bioprosthetic aortic valve,
periodontal disease
None
Mi prolapse
Aortic insufficiency, scalp wound

3/56/m
4/68/m
5/47/m
6/62/f
7/74/m
8/42/m

Therapy

Valve
replacement

Spondylodiscitis, cure

11, 1982

Ao

Yes

Phlebitis, cure

11, 1982

Mi
Ao
Ao

Benzylpenicillin, gentamicin
Benzylpenicillin, streptomycin
Erythromycin, rifampin

No
Yes
Yes

Cure
Cure
Cure

11, 1982
31, 1984
39, 1985

Mi
Mi
Aob

Benzylpenicillin, tobramycin, clindamycin

Benzylpenicillin, gentamicin, amoxicillin
Vancomycin, fusidic acid, amoxicillin,
gentamicin
Amoxicillin 1 clavulanic acid, gentamicin
Benzylpenicillin, gentamicin
Benzylpenicillin, gentamicin

No
No
No

Cure
Cure
Cure

28, 1989
8, 1991
22, 1993

No
Yes
Yes

Cure
Cure
Right dorsalis pediculis
territory embolus, cure
Allergic neutropenia due to
cefuroxim, cure

25, 1993
15, 1993
34, 1994

Kidney abscess, cure

Patient 1

12/34/m Multiple valve replacements with

prosthetic valve, infective
endocarditis, asymptomatic dental
sepsis, benzylpenicillin allergy
13/63/m Chronic obstructive bronchitis, poor
dentition

Ao

Cefuroxime, tobramycin, ciprofloxacin,

erythromycin

No

Mi

Amoxicillin, amikacin

Yes

43, 1995

Abbreviations: Ao, aortic valve; Mi, mitral valve; Tri, tricuspid valve; NA, not available; m, male; f, female.
No evidence of vegetation on echocardiogram.

fied, provided that the genes of closely related species share a

significantly lower degree of similarity. However, if this is not
the case, complete identification can be achieved in two ways.
First, confirmation can be achieved by a few simple additional

FIG. 2. Examples of the usefulness of vancomycin test to determine the

Gram reaction for gram-variable bacteria. The vancomycin-susceptible grampositive bacterium is G. haemolysans (A), and the vancomycin-resistant gramnegative bacteria are Acinetobacter (B), Moraxella sp. (C), and Kingella kingae
(D). Arrowheads indicate the limits of the inhibition zone around vancomycin
(VA) and colistin (CS) disks.

biochemical tests, as described for patient 3 of this study. This

method is quick and cheap, but it requires that a bacterium be
easily subcultured. If this is not possible, additional sequencing
can be used in order to determine a complete 16S rRNA gene
sequence. This second approach is more expensive and timeconsuming, but it avoids the need for additional phenotypic or
specific antibody tests, both of which may not be routinely
available in the diagnostic laboratory and thus would require
referral to a reference laboratory. Furthermore, this approach
also allows the description of new pathogens (16) which may
have been misidentified by standard phenotypic identification
schemes. Finally, the use of this technique for identification
does not require an experienced microbiologist and gives a
universal bacterial identification ability to all laboratories, provided that they are equipped with an automated sequencer.
With increasing availability and decreasing costs, such equipment is likely to become a feature of more and more routine
laboratories in the years to come.
However, for the present, phenotypic characterization remains the standard approach to bacterial identification, with
Gram staining being one of the most important first steps of
most routine identification schemes (5). A mistake at this stage
can lead to the application of inappropriate tests and therefore
unnecessary delays in processing; thus, it is important that all
efforts be made to ensure correct interpretation of the Gram
staining result. The reference method for assessing cell wall
type is electron microscopy, and although it is accurate, the
method is expensive, is time-consuming, and requires specialized equipment. As an alternative we have added vancomycin
and colistin to our standard antibiotic susceptibility tests. Most
gram-positive bacteria are susceptible to vancomycin, whereas
most gram-negative bacteria are susceptible to colistin. Such a
method has previously been shown to be beneficial for the
determination of cell wall type among nonenterobacterial rods
(24). The test is easy to perform (Fig. 2) and is inexpensive. It
must be noted, however, that this test is not definitive, since a

Downloaded from http://jcm.asm.org/ on June 9, 2015 by guest

No

Mi
Mi
Ao

Reference,
year

Cefamandole, gentamicin, benzylpenicillin,

amoxicillin
Benzylpenicillin, gentamicin

9/71/m Colonic carcinoma

10/53/f Mi regurgitation
11/20/m None

Outcome

MOLECULAR IDENTIFICATION OF GEMELLA SPECIES

VOL. 36, 1998

vancomycin-resistant strain of G. haemolysans has been encountered (18).

In conclusion, for bacteria which are easily identified by
phenotypic schemes (especially with the help of commercial
identification kits or simple additional tests) and which represent more than 99.9% of the bacteria isolated in clinical microbiology laboratories, no additional identification technique
is required. However, 16S rRNA gene analysis is becoming
more competitive in terms of efficiency and accuracy for fastidious bacteria or those not easily identified by phenotypic
schemes. The use of 16S rRNA gene analysis should also lead
to an increase in the number of descriptions of new pathogens
and in the recovery of unexpected pathogens.

22.
23.
24.
25.
26.
27.

ACKNOWLEDGMENT
We thank Richard Birtles for correcting the manuscript.

1. Abboud, R., and A. Friart. 1995. Deux cas dendocardite tricuspidienne

isolee apre`s intervention colique. Acta Clin. Belg. 50:242245.
2. Acar, J. F., and F. W. Goldstein. 1996. Disk susceptibility test, p. 151. In V.
Lorian (ed.), Antibiotics in laboratory medecine, 4th ed. The Williams &
Wilkins Co., Baltimore, Md.
3. Appelbaum, P. C., M. R. Jacobs, J. L. Heald, W. M. Palko, A. Duffett, R.
Crist, and P. A. Naugle. 1984. Comparative evaluation of the API 20S system
and the AutoMicrobic System Gram-Positive Identification Card for species
identification of streptococci. J. Clin. Microbiol. 19:164168.
4. Aspevall, O., E. Hillebrandt, B. Linderoth, and M. Rylander. 1991. Meningitis due to Gemella haemolysans after neurosurgical treatment of trigeminal
neuralgia. Scand. J. Infect. Dis. 23:503505.
5. Baron, E. J., A. S. Weissfeld, P. A. Fuselier, and D. J. Brenner. 1995.
Classification and identification of bacteria, p. 249264. In P. R. Murray, E. J.
Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.), Manual of
clinical microbiology, 6th ed. American Society for Microbiology, Washington, D.C.
6. Bell, E., and A. C. McCartney. 1992. Gemella morbillorum in an intravenous
drug abuser. J. Infect. 25:110112.
7. Berger, U. 1961. A proposed new genus of gram negative cocci: Gemella. Int.
Bull. Bacteriol. Nomencl. Taxon. 11:1719.
8. Brack, M. J., P. G. Avery, P. J. B. Hubner, and R. A. Swann. 1991. Gemella
haemolysans: a rare and unusual cause of infective endocarditis. Postgrad.
Med. J. 67:210212.
9. Buu-Hoi, A., A. Sapoetra, C. Branger, and J. F. Acar. 1982. Antimicrobial
susceptibility of Gemella haemolysans isolated from patients with subacute
endocarditis. Eur. J. Clin. Microbiol. 1:102106.
10. Calopa, M., F. Rubio, M. Aguilar, and J. Peres. 1990. Giant basilar aneurysm
in the course of subacute bacterial endocarditis. Stroke 21:16251627.
11. Chatelain, R., J. Croize, P. Rouge, C. Massot, H. Dabernat, J. C. Auvergnat,
A. Buu-Hoi, J. P. Stahl, and F. Bimet. 1982. Isolement de Gemella haemolysans dans trois cas dendocardites bacteriennes. Med. Mal. Infect. 1:2530.
12. Coto, U., and S. L. Berk. 1984. Endocarditis caused by Streptococcus morbillorum. Am. J. Med. Sci. 287:5457.
13. Debast, S. B., R. Koot, and J. F. Meis. 1993. Infections caused by Gemella
morbillorum. Lancet 342:560.
14. Dessen, P., C. Frondat, C. Valencien, and G. Munier. 1990. BISANCE: a
French service for access to biomolecular sequences databases. Comput.
Appl. Biosci. 6:355356.
15. Devuyst, O., P. Hainaut, J. Gigi, and G. Wauters. 1993. Rarity and potential
severity of Gemella haemolysans endocarditis. Acta Clin. Belg. 48:5253.
16. Drancourt, M., L. Niel, and D. Raoult. 1995. Unknown multiresistant gram
negative bacterium causing meningitis in HIV-positive patient in Africa.
Lancet 346:1168.
17. Durack, D. T., E. L. Kaplan, and A. L. Bisno. 1983. Apparent failures of
endocarditis prophylaxis. JAMA 250:23182322.
18. Efstratiou, A., D. Morrisson, and N. Woodford. 1993. Glycopeptide-resistant
Gemella haemolysans from blood. Lancet 342:927928.
19. Facklam, R. R. 1977. Physiological differentiation of viridans streptococci.
J. Clin. Microbiol. 19:184201.
20. Facklam, R. R., D. L. Rhoden, and P. B. Smith. 1984. Evaluation of the
Rapid Strep system for the identification of clinical isolates of Streptococcus
species. J. Clin. Microbiol. 20:894898.
21. Freney, J., S. Bland, J. Etienne, M. Desmonceaux, J. M. Boeufgras, and J.

29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.

45.
46.
47.
48.
49.
50.

Fleurette. 1992. Description and evaluation of the semiautomated 4-hour

Rapid ID 32 Strep method for identification of streptococci and members of
related genera. J. Clin. Microbiol. 30:26572661.
Fresard, A., V. P. Michel, X. Rueda, G. Aubert, G. Dorche, and F. Lucht.
1993. Gemella haemolysans endocarditis. Clin. Infect. Dis. 16:586587.
Garavelli, P. L. 1990. Meningite da Streptococcus morbillorum. Minerva
Med. 81:69.
Graevenitz, A., and C. Bucher. 1983. Accuracy of the KOH and vancomycin
tests in determining the Gram reaction of nonenterobacterial rods. J. Clin.
Microbiol. 18:983985.
Helft, G., X. Tabone, J. P. Metzger, and A. Vacheron. 1993. Gemella haemolysans endocarditis with colonic carcinoma. Eur. J. Med. 2:369370.
Holdeman, L. V., and W. E. C. Moore. 1974. New genus Coprococcus, twelve
new species, and emended description of four previously described species
from human feces. Int. J. Syst. Bacteriol. 24:260277.
Joblet, C., V. Roux, M. Drancourt, J. Gouvernet, and D. Raoult. 1995.
Identification of Bartonella (Rochalimea) species among fastidious gramnegative bacteria on the basis of partial sequence of the citrate-synthase
gene. J. Clin. Microbiol. 33:18791883.
Kaufhold, A., D. Franzen, and R. Lu
tticken. 1989. Endocarditis caused by
Gemella haemolysans. Infection 17:385387.
Kerr, J. R., C. H. Webb, J. G. McGimpsey, and N. P. S. Campbell. 1994.
Infective endocarditis due to Gemella morbillorum complicating hypertrophic obstructive cardiomyopathy. Ulster Med. J. 63:108110.
Kilpper-Ba
lz, R., and K. H. Schleifer. 1988. Transfer of Streptococcus morbillorum to the genus Gemella as Gemella morbillorum, comb. nov. Int. J.
Syst. Bacteriol. 38:442443.
Laudat, P., P. Cosnay, B. Icole, A. Raoult, P. Raynaud, and M. Brochier.
1984. Endocardite `a Gemella haemolysans: une nouvelle observation. Med.
Mal. Infect. 4:159161.
Ludwig, W., M. Weizenegger, R. Kilpper-Ba
lz, and K. H. Schleifer. 1988.
Phylogenetic relationships of anaerobic streptococci. Int. J. Syst. Bacteriol.
38:1518.
Martin, M. J., D. A. Wright, and A. R. R. Jones. 1995. A case of Gemella
morbillorum endocarditis. Postgrad. Med. J. 71:188.
Matsis, P. P., and R. N. Easthorpe. 1994. Gemella haemolysans endocarditis.
Aust. N. Z. J. Med. 24:417418.
Maxwell, S. 1989. Endocarditis due to Streptococcus morbillorum. J. Infect.
18:6772.
May, T., C. Amiel, C. Lion, M. Weber, A. Gerard, and P. Canton. 1993.
Meningitis due to Gemella haemolysans. Eur. J. Clin. Microbiol. Infect. Dis.
12:644645.
Miller, L., and T. Berger. 1985. Bacterial identification by gas chromatography of whole cell fatty acids. Hewlett-Packard application note 228241.
Hewlett-Packard, Avondale, Pa.
Mitchell, R. G., and P. J. Teddy. 1985. Meningitis due to Gemella haemolysans after radiofrequency trigeminal rhizotomy. J. Clin. Pathol. 38:558560.
Morea, P., M. Toni, M. Bressan, and P. Stritoni. 1991. Prosthetic valve
endocarditis by Gemella haemolysans. Infection 19:446.
Omran, Y., and C. A. Wood. 1993. Endovascular infection and septic arthritis
caused by Gemella morbillorum. Diagn. Microbiol. Infect. Dis. 16:131134.
Prevot, A. R. 1933. Etudes de sytematique bacterienne. I. Lois generals. II.
Cocci anaerobies. Ann. Sci. Nat. 15:23260.
Ruoff, K. L. 1990. Gemellae: a tale of two new species (and five genera). Clin.
Microbiol. Newsl. 12:14.
Samuel, L., and P. Bloomfield. 1995. Gemella haemolysans prosthetic valve
endocarditis. Postgrad. Med. J. 71:188.
Smith, L. 1957. Peptostreptococcus Kluyver and van Niel (1936), p. 533541.
In R. S. Breed, E. G. D. Murray, and N. R. Smith (ed.), Bergeys manual of
determinative bacteriology, 7th ed. The Williams & Wilkins Co., Baltimore,
Md.
Terada, H., K. Miyahara, H. Sohara, M. Sonoda, H. Venomachi, J. Sanada,
and T. Arima. 1994. Infective endocarditis caused by an indigenous bacterium (Gemella morbillorum). Intern. Med. 33:628631.
Thjo
ta, T., and J. Boe. 1938. Neisseria haemolysans. A haemolytic species of
Neisseria Treviscan. Acta Pathol. Microbiol. Scand. Suppl. 37:527531.
Tunnicliff, R. 1917. The cultivation of a micrococcus from blood in preeruptive and eruptive stage of measles. JAMA 68:10281030.
Von Essen, R., M. Ika
valko, and B. Forsblom. 1993. Isolation of Gemella
morbillorum from joint fluid. Lancet 342:177178.
Weisburg, W. G., S. M. Barns, D. A. Pelletier, and D. J. Lane. 1991. 16S
ribosomal DNA amplification for phylogenic study. J. Bacteriol. 173:697
703.
Whitney, A. M., and S. P. OConnor. 1993. Phylogenetic relationship of
Gemella morbillorum to Gemella haemolysans. Int. J. Syst. Bacteriol. 43:832
838.

Downloaded from http://jcm.asm.org/ on June 9, 2015 by guest

REFERENCES

28.

871