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Vol. 36, No. 3
INTERNATIONAL
JOURNAL OF SYSTEMATIC
BACTERIOLOGY,
July 1986, p. 431434
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Copyright 0 1986, International Union of Microbiological Societies
Phenotypic and Genotypic Comparisons among Strains of the
Lobster Pathogen Aerococcus viridans and Other Marine
Aerococcus viridans-Like Cocci
R. WIIK,’* V. TORSVIK,’ AND E. EGIDIUS’
Institute of Marine Research, Directorate of Fisheries, 501 1 Bergen-Nordnes, and Department of Microbiology and Plant
Physiology, University of Bergen, N-5014 Bergen- University,’ Norway
’
The lobster pathogen Aerococcus viridans and other gram-positive, marine A. viridans-like cocci were
examined morphologically, biochemically, and genetically. Morphologically, the lobster pathogenic strains
were unique in their tetrad-forming capacity. Because of intragroup fermentative variations among the
lobster-pathogenic strains and values of deoxyribonucleic acid (DNA) base composition overlapping those of the
other cocci, the lobster pathogens did not stand out as a separate group based on these data. According to the
DNA-DNA hybridization studies, however, the lobster-pathogenic strains were very closely related (80 to 100%
homology) and could be easily separated from the remaining strains. All the cocci had extraordinarily small
genomes ranging from 0.57 X lo9 to 1.01 X lo9 daltons.
The lobster pathogen Aerococcus viridans (8), originally
“ G a r n u homari” (12), is a gram-positive tetracoccus which
causes fatal septicemia (gaffkemia) in lobsters. There has
been considerable disagreement about the taxonomic relationship of A . viridans and “ G . homari.” A . viridans strains
initially included cocci isolated from air (22). Deibel and
Niven ( 5 ) proposed to include both the air cocci and the
lobster pathogens in the genus Pediococcus, although subsequent deoxyribonucleic acid (DNA) homology studies (18,
19) did not support a close relationship to this genus. Kelly
and Evans (14) examined DNA-DNA homology among
lobster-pathogenic strains and nonmarine strains of A .
viridans and found strong evidence that both groups belong
to the same species; this designation was introduced in the
8th edition of Bergey ’s Manual of Determinative Bacteriology (8). Kelly and Evans (14) also suggested that it might be
desirable to separate the lobster-pathogenic strains from
avirulent strains of A . viridans by considering the lobster
pathogen a subspecies. In the present text we generally refer
to strains of A , viridans originating from gaflkemic lobsters
as A . viridans GL.
The present study compared the morphology, physiology,
and genetic features of A . viridans GL strains, isolated from
two species of lobsters (Homarus gammarus L. and H .
americanus H. Milne Edwards 1837), and of representative
avirulent strains of A . viridans-like cocci of marine origin.
Since most earlier genomic studies of A . viridans GL examined only isolates from the lobster H . americanus (14, 18,
19), the present study provides additional data on the biology
and genetic characteristics, including DNA base composition, genome size, and ,DNA-DNA hybridization comparisons, among other lobster strains.
MATERIALS AND METHODS
Bacterial strains. A . viridans GL strains HI520, HI510,
and SH21 were isolated from Norwegian H. gammarus;
STA.14, STA.18, and Rabin were from H . americanus (J. E.
Stewart, Fisheries and Oceans, Resource Branch, Nova
Scotia, Canada); ATCC 10400 was from H . americanus
(American Type Culture Collection, Rockville, Md.);
* Corresponding author.
NCMB 1120 was from British H . gammarus (National
Collection of Marine Bacteria, Aberdeen, Scotland); and A .
viridans strain NCTC 8251T (T indicates type strain) was
from air (National Collection of Type Cultures, Colindale,
England). A . viridans GL strain KB161 and the unidentified
marine strains KB162, KB172, SH22, SH23, and SH31 were
isolated from water or sediment in Norwegian lobster ponds.
The marine isolates 37R and 88A were supposed to be
avirulent strains of A . viridans (J. E. Stewart). Escherichia
coli B (ATCC 11303) was used as a reference strain in the
DNA studies.
Morphological and biochemical characteristics. Capsule
staining was done by the method of Collins (3). Bacterial
cells were grown at 30°C for 24 h on nutrient agar (Oxoid
Ltd., London, England) supplemented with 5% human
blood. This temperature and incubation period were generally used throughout the study as standard growth conditions.
The Hucker modification of Gram stain (4) was used on
cells grown on brain heart infusion agar (BHIA) (Difco
Laboratories, Detroit, Mich.). Cell morphology and motility
were determined microscopically on cultures grown in tryptone soya broth (TSB) (Oxoid). Stability of tetrad formation
was tested by ultrasonic treatment (MSE-Mullard Ultrasonic
Disintegrator, MSE Scientific Instruments Ltd., Crawley,
West Sussex, England) of 5-ml TSB cultures of A . viridans
GL strains for 5 , 10, 20, 30, 60, and 120 s . Each culture was
studied microscopically and counted with a counting chamber (Hawksley and Sons Ltd., Lansing, West Sussex, England) before and after treatment. Hemolysis was tested by
growing the bacteria for 48 h on nutrient agar containing 5%
defibrinated sheep blood. Catalase production was detected
by transferring bacterial cells grown on BHIA to 1 drop of
3% H202solution. Acid production was measured by growing the organisms in a medium containing 0.65% glucose,
0.45% yeast extract (Oxoid), 1.5% tryptone (Oxoid), 0.64%
NaCl, 0.25% phenyl ethyl alcohol, and 0.0008%bromcresol
purple (wthol) (pH 7.4) (21). pH readings were taken after 24
h. The fermentative ability of the organisms was determined
by the API 50 CHE system (API System SA, France). The
bacteria were grown on tryptone soya agar (Oxoid) before
transfer to the API 50 CHE medium. Readings were taken
after 48 h.
431
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INT. J . SYST.BACTERIOL.
WIIK ET AL.
Virulence tests. Bacterial strains were grown in TSB and
diluted in 3% NaCl to concentrations from 6 x lo1 to 2.6 x
lo4 CFU/ml; 0.5 ml of the suspension was injected into the
lobsters, each of which weighed approximately 0.5 kg and
had been shown to be free of the pathogen by the method of
Stewart et al. (21) before being included in the experiment.
In moribund or newly dead lobsters, gaffkemia was easily
recognized by microscopic appearance of numerous grampositive tetrads in the hemolymph.
DNA extraction and purification. Strains were grown in
TSB for 17 to 30 h, and cells were harvested just before the
stationary growth phase. E. coli B was cultivated in a
medium containing 0.3% meat extract (Difco), 0.5% peptone
(Oxoid), and 0.2% yeast extract (Oxoid) dissolved in distilled
water (wt/vol) at 37°C for 15 h. The cells were harvested and
lysed, and the nucleic acids were extracted and purified by
the method of Marmur (16), with two modifications: immediately after adding sodium lauryl sulfate, the lysozymetreated cell mixtures became viscous and nearly crystal
clear, and the prescribed incubation at 60°C was therefore
omitted; and the DNA from the cocci was easily spooled
onto a glass rod after mild shaking of the mixture by hand.
Ratios of absorbance at 260 nm (A260)/A280 and A260/A230
were used to assess DNA purity.
Guanine plus cytosine (G+C) content of DNA was determined by thermal denaturation (15), with 0 . 5 ~standard
saline citrate (SSC) ( l x SSC is 0.15 M NaCl plus 0.015 M
sodium citrate) as the solvent and DNA at concentrations
corresponding to initial A260 of 0.4 to 0.7, representing 20 to
35 pg of DNA per ml(l5). A Pye Unicam spectrophotometer
connected to a microprocessor (Rockwell, AIM 65,
MicroNor) was used in the DNA studies. Moles percent
(mol%) G + C was calculated by the equation: mol% G+C =
( T , - 69.37 - 15.2 log 0.5)/0.41, where T,,, is melting
temperature (10). Hyperchromicity was calculated as the
percent increase in A260.
Determination of M. The molecular weight of the bacterial
genome (M) was determined based on initial renaturation
rate of fragmented DNA, recorded spectrophotometrically
(11). The renaturation temperatures of DNA from the cocci
and E. coli B were 64.5 and 72”C, respectively. The A260 of
sheared DNA in the renaturation solution was 0.56 at 22”C,
equivalent to a DNA concentration of 28 pglml (15). The
molecular weight of the bacterial genome was calculated by
the equation: M = (70.03 - 0.35 x mol% G+C) x 107/k’ (9),
where k’ represents the reaction rate constant.
DNA-DNA hybridization. Determination of the genetic
relationship between two bacterial strains was based on
initial renaturation rates of the DNA types and their mixture,
recorded spectrophotometrically (6). The percentage of hybridized DNA was calculated by equation 19 of De Ley et al.
(6). Two modifications of the methods of De Ley et al. (6)
and Gillis et al. (10) were used: spectrophotometric instead
of chemical determination of DNA concentration in the
renaturation solution, and decrease of DNA concentration in
the renaturation solution from a 80 to 28 Fg/ml (A260 = 0.56).
As the value of k’ for E . coli B DNA is in agreement with the
values given by Gillis et al. (9, lo), determining the DNA
concentration spectrophotometrically instead of chemically
and decreasing the concentration from 80 to 28 pg of DNA
per ml seems justifiable. The genome size of E. coli B is also
in agreement with figures given by Bak et al. (1) and Cairns
(2). Huss et al. (13) recommended that a concentration of 30
to 40 pg of DNA per ml be used in the method developed by
De Ley et al. (6). Four to six replicates per DNA type
mixture were used, and reproducibility was obtained with an
TABLE 1. Virulence of A . vividuns GL strains tested by lobster
infection experiments
Strain
No. of
lobsters
infected
ATCC 10400
2
HI520
2
SH21
2
KB161
HI510
1
1
CFU injected
Water temp
(“‘I
18.8 +18.2 +18.8 ?
18.2 k
18.7 2
18.3 2
18.0 2
12.0 2
1.2”
1.4
2.0
2.2
1.4
1.1
2.0
1.0
(2.17 2
(2.60 5
(1.31 ?
(1.53 k
(9.75 ?
(3.50 &
(5.60 2
(3.00 2
0.19’)
0.21)
0.05)
0.05)
0.79)
0.30)
0.41)
0.19)
x 10’
x 103
X lo4
x 10’
x lo2
x lo2
x lo3
x 10’
Time to
death
(h)
7
7
5
5
7
11
Average deviation from the average temperature.
Standard error (11).
accuracy for change in A260of 2 5 x
represents v’ 5 1.88 X lop5.
(k0.25 mm), which
RESULTS
All bacterial strains examined were nonmotile, gram positive, alpha-hemolytic, catalase positive, and acid producing.
For the A . viridans GL strains, the pH of the medium after
growth, which stabilized after 24 h, varied from 5.5 to 5.8,
and for the remaining strains, the pH ranged from 4.2 to 5.4.
Only the A . viridans GL strains formed tetrads. The other
strains were diplococci, except for SH23, which formed
slightly ovoid cells in pairs and chains. Before ultrasonic
treatment, A . viridans GL occurred in tetrads and in clusters
of numerous cells. After 20 s of ultrasonic treatment, only
tetrads could be seen under the microscope, and the number
of cell units had increased 17 times, indicating that each
cluster contained an average of 17 tetrads. After 120 s of
treatment many of the tetrads had divided into diplococci,
and the number of cell units had increased by a factor of 28.
Only strains of A . viridans GL appeared to form capsules on
blood agar. In the API 50 CH tests, A . viridans GL strains
gave the same general reaction pattern, fermenting galactose, D-glucose, D-fructose, D-mannose, mannitol, N acetylglucosamine, amygdalin, arbutin, esculin, cellobiose,
maltose, lactose, saccharose, trehalose, inulin, D-raffinose,
P-gentibiose, gluconate, and giving a rather weak positive
fermentation of ribose. The strains showed intragroup variation with respect to fermentation of sorbitol, a-methyl-Dglucoside, melibiose, D-turanose, and 5-ketogluconate. A .
viridans NCTC 8251T differed from the lobster-derived
strains in inability to ferment trehalose, inulin, D-raffinose,
p-gentibiose, and gluconate, and by demonstrating only
weak positive fermentat i o n of N - acet y lglu c o s ami n e ,
amygdalin, arbutin, and esculin. The avirulent A . viridans
strains also presented some differences in fermentative patterns: strain 37R did not ferment ribose or inulin, and strain
88A was not able to catabolize ribose, inulin, gluconate,
mannitol, or amygdalin. In addition, the other marine, A .
viridans-like cocci displayed fermentative patterns that differed from those of A . viridans G L isolates: strains KB162
and KB172 both failed to ferment inulin, amygdalin, arbutin,
esculin, cellobiose, trehalose, P-gentibiose, or gluconate;
strain SH22 did not ferment mannitol, trehalose, or gluconate, but D-lyxose, D-fucose, and L-fucose; strain SH23 did
not ferment galactose, lactose, trehalose, inulin, D-raffinose,
or gluconate; and strain SH31 failed to ferment ribose,
galactose, P-gentibiose, or gluconate.
The limited number of lobsters available excluded virulence tests on all bacterial strains in this study. Of those
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VOL.36, 1986
LOBSTER PATHOGEN A . VZRIDANS
TABLE 2. DNA base composition and genome size of various A .
viriduns and A . viriduns-like strains
G+CQ
Mol% Hyperchromicity
(%)
kt
M (109
daltons)b
E . coli B
52.2
34.0
0.192
2.70
A . viriduns GL
HI520
HI510
KB161
SH21
Rabin
STA. 14
STA.18
ATCC 10400
NCMB 1120
38.8
38.8
38.8
39.8
38.3
38.8
39.8
37.6
39.0
34.1
35.9
27.4
36.9
28.7
28.4
33.8
30.8
26.3
0.804
0.804
0.877
0.765
0.785
0.804
0.731
0.824
0.70
0.70
0.64
0.73
0.71
0.70
0.77
0.69
A . viriduns (avirulent)
37R
88A
39.3
39.8
30.4
35.4
0.692
0.987
Strain
Unidentified marine isolates
( A . viriduns-like)
KB162
KB172
SH22
SH23
SH31
34.7
31.5
34.4
39.2
34.0
39.8
40.0
37.3
37.1
39.5
0.81
0.63
0.984
0.565
0.57
1.01
0.897
0.63
The values of DNA base composition (mol% G + C) and hyperchromicity
were determined by thermal denaturation in 0.5 x SSC.
M was determined from the DNA renaturation rate, measured
spectrophotometrically; k', apparent renaturation rate constant.
~~
~~~
~~~~~~~~
~
433
TABLE 3. DNA-DNA hybridization among various A . viridans
GL isolates and other A . viriduns and A . viridans-like strains
Strains used for recipient DNA
A . viriduns GL
HI520
HI510
KB161
SH21
Rabin
STA. 14
STA.18
ATCC 10400
A . viriduns (avirulent)
37R
88A
Unidentified marine isolates
( A , viriduns-like)
KB172
SH22
SH31
Donor DNA" prepared from:
HI520
100
100
87.
86
93
96
80
84
KB161
SH21
86
100
86
86
100
56
48
38
26
1
33
31
a Percent hybridized DNA, determined spectrophotometrically, from initial
renaturation rates. Values are normalized to 100% for homologous
combinations.
~
a
examined, only strain ATCC 10400 was found to be avirulent
(Table 1).
The A26dA280ratio of DNA ranged from 1.8 to 2.0, with a
mean value of 1.8. The A26dA230
ratio ranged from 1.6 to 1.8
with a mean of 1.7 for the A . viriduns G L strains plus 37R
and 88A, and from 1.9 to 2.2 with a mean of 2.1 for the
unidentified strains and for E. coli B.
The G+C values of DNA are given in Table 2. By melting
the DNAs several times, an accuracy of k0.2 mol% G + C
could be estimated. For E . coli B DNA, G+C was 52.2
mol%, which is in accordance with previously found G + C
values (10; K. Salte, Ph.D thesis, University of Bergen,
Norway, 1981). DNA from A . viriduns ATCC 10400 had
G + C of 37.6 mol%, which is in accordance with previous
results (14, 18, 19).
In addition to the absorbance ratios, the hyperchromicity
values indicated pure DNA (lo), as did the regularity of
thermal melting profiles.
Molecular weights of the bacterial genomes are given in
Table 2. The mean 2 standard deviation for A . viriduns G L
is (0.71 +. 0.04) x lo9.
The DNA-DNA hybridizations showed a high degree of
relatedness (80 to 100% hybridized DNA) among the strains
of A . viriduns GL; the relatedness between A . viriduns GL
and the remaining strains varied from 1 to 56% (Table 3). At
similarities below 25 to 30%, hybridized DNA values calculated by equation 19 of De Ley et al. (6) are less reliable and
give only semiquantitative values (6, 13).
DISCUSSION
The nonmarine strain A . viriduns NCTC 8251, which was
originally described by Williams et al. (22), deviated from the
lobster-pathogenic group in five fermentative tests, and by
giving a sparse, granular suspension of growth in TSB.
Because of variation in fermentative ability, both among
nonmarine strains of A . viriduns (22) and lobster-pathogenic
strains, fermentative testing does not clearly differentiate
between these two groups. A prominent phenotypic characteristic of the A . viriduns G L strains was their tetrad-forming
capacity, not found in other strains in the present study.
Nonmarine strains of A . viriduns may also form packets of
but according to Williams et al. (22) they normally
four (3,
occur in pairs or irregular clusters.
Serological grouping of virulent and avirulent strains of A .
viriduns indicates that virulent strains possess a special
antigen absent from avirulent strains (20). Steenbergen et al.
(20) emphasize, however, that the presence or absence of the
virulence antigen is not in itself justification for dividing the
organisms into two taxonomic groups. By injection in lobster, strain ATCC 10400 appeared to be avirulent. Earlier,
this strain was shown to be pathogenic to both H . gammurus
(7) and H . americunus (12). This loss of virulence was not
reflected in morphologic, biochemical, or genetic changes,
when compared to corresponding characteristics of virulent
strains.
The G + C values of DNA obtained for the A . viriduns GL
strains overlap those previously found for nonmarine strains
of A . viriduns (14, 18, 19). Our DNA-DNA hybridization
results showed a very close relationship among the lobsterpathogenic strains. In a previous DNA-DNA hybridization
study, a comparatively high degree of homology between
lobster-pathogenic and nonmarine A . viriduns was observed
(14). In contrast to the very high degree of homology among
the lobster-pathogenic strains, however, the nonmarine
aerococci showed a rather large intragroup variation.
According to our DNA-DNA hybridization results, the
strains 37R, 88A, KB172, SH22, and SH31 hardly belong to
the species A . viridans, represented by the lobsterpathogenic group. The different homology values between
reference strain HI 520 and each of the unidentified strains
may reflect a rather distant relationship among the unidentified strains. The API 50 CH tests, however, did not give a
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434
INT. J. SYST.BACTERIOL.
WIIK ET AL.
satisfactory separation of the bacterial groups or strains. As
an example, strain 37R deviated from the lobster pathogenic
group, which in itself varied with respect to five traits, solely
in its inability to ferment ribose and inulin, while the
avirulent A. viridans strain NCTC 8251 deviated from the
lobster pathogens in five different fermentative traits. The
fermentative variations indicate, however, that these strains
probably belong to several different groups. Nor did determination of DNA base composition give a clear differentiation between these strains, exhibiting G + C values ranging
from 37.1 to 40.0 mol%. According to Schultes and Evans
(19), A. viridans species represents G + C values ranging
from 37.0 to 40.2 mol%.
The A. viridans GL strains examined here have remarkably small genomes, ranging from 0.57 X lo9 to 1.01 X lo9
daltons. Organisms in the class Mollicutes (mycoplasmas)
which represent the smallest genomes previously recorded
for procaryotes, have genome sizes of 0.5 x lo9 1.0 X lo9
daltons (17). Bak et al. (1)determined bacterial genome sizes
by DNA-DNA renaturation studies and found molecular
weights ranging from 1.0 X lo9 to 7.0 x lo9; selected
members of the family Micrococcaceae had genome sizes
from 1.12 x lo9 to 2.82 x lo9 daltons, and those in the genus
Streptococcus had genome sizes from 1.20 x lo9 to 1.37 x
lo9 daltons. Haemophilus influenzae and Neisseria catarrhalis had the smallest genomes (1.01 x lo9 and 1.04 x lo9
daltons, respectively), and Pseudomonas aeruginosa had
the largest (6.96 x lo9 daltons). Based on DNA-DNA
renaturation rates, Gillis and De Ley (9) determined the
genome sizes of 40 different bacterial strains ranging from
1.40 X lo9 to 3.99 x lo9 daltons, the extremes represented
by Nitrosomonas sp. and Serratia marcescens, respectively.
The very small genomes obtained for the bacteria in the
present study show that genomes smaller than 1.0 x lo9
daltons also exist among procaryotes other than the
mycoplasmas .
ACKNOWLEDGMENTS
We thank J. Goksgyr? K. Salte, and K. A. Hoff for their support
during this study, and A. S . Lyssand for her secretarial assistance.
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