588 Article Vol. 9, No.

8
Eur. J. Clin. Microbiol. Infect Dis., August 1990,p. 588-594
0934-9723/90/08 0588-07 $ 3.00/0

Typing of Coagulase-Negative Staphylococci by

Southern Hybridization of Chromosomal DNA
Fingerprints Using a Ribosomal RNA Probe

H. Bialkowska-Hobrzanska 1' 2', V. Harry 1, D. Jaskot 1, O. H a m m e r b e r g z' 2

Ribotyping consists of restriction endonuclease fingerprinting of ribosomal RNA

(rRNA) genes visualized by Southern hybridization with an rRNA probe. This
method was developed and compared with restriction endonuclease fingerprinting
of chromosomal D N A for typing coagulase-negative staphylococci. Twenty-five
American Type Culture Collection reference type strains and 53 clinical isolates
were typed. Both methods clearly distinguished all 15 species of coagulase-negative
staphylococci and most individual strains within each species. Except in the case of
Staphylococcus warneri, ribotyping was most discriminating with the use of ClaI,
one of eight endonucleases tested. HpaI and AvaI were more specific than Clal for
discrimination between strains of Staphylococcus warneri. The patterns produced
by ribotyping were much simpler and thus easier to interpret than corresponding
chromosomal fingerprints. However, ribotyping was slightly less discriminating. It
is concluded that ribotyping offers an alternative method for molecular typing of
coagulase-negative staphylococci. The application of both methods needs to be
further evaluated in the clinical setting.

Over the past decade coagulase-negative restriction endonuclease fingerprinting of chro-

staphylococci have emerged as important
pathogens responsible for hospital-acquired
bacteremia in neonatal intensive care units
(1, 2). Efforts to identify the source of these
infections and modes of their transmission
within the hospital have been hampered by the
lack of sufficiently discriminatory typing
systems which could distinguish between strains
colonizing skin and mucosal surfaces and those
causing invasive disease.
Several molecular typing techniques have been
applied to studying the epidemiology of co-
agulase-negative staphylococci infections. Plas-
mid D N A analysis has been shown to be of
limited value since it is restricted to strains that
carry more than one plasmid, and occasionally
plasmid content is unstable and therefore
unsuitable for epidemiological investigations
(3, 4). Chromosome typing, also known as
1Department of Microbiology and Infectious Diseases,
St. Joseph's Health Centre, 268 Grosvenor Street,
London, Ontario, Canada N6A 4V2.
2Department of Microbiology and Immunology, University
of Western Ontario, London, Ontario, Canada N6A 5C1.
mosomal DNA, has been applied to discrimina-
tion of strains of Staphylococcus epidermidis
and Staphylococcus haemolyticus and has been
found more specific and reproducible than
plasmid analysis (4). A major advantage of
chromosome typing is that it examines the
chromosome, which is more stable than the
plasmid content. A disadvantage is that com-
paring electrophoretic patterns consisting of up
to 100 bands is difficult to interpret. Without the
use of sophisticated analytical methods, such as
scanning devices incorporating pattern-match-
ing software, chromosome typing is unlikely to
be widely applicable for typing large numbers of
strains.
Recent advances in molecular typing consist of
the use of nucleic acid probes to highlight
restriction site heterogeneity within selected
areas of bacterial chromosome (5-8). One
approach, ribotyping, compares restriction site
heterogeneity within rRNA genes and their
adjacent regions. This approach employs
restriction endonuclease fingerprinting of chro-
mosomal DNA and Southern hybridization with
the use of an rRNA probe. Ribosomal R N A
operons, which encode 23S, 16S and 5S rRNA,
.Vol. 9, 1990 589

e x i s t in a h i g h c o p y n u m b e r in a v a r i e t y o f infants with septicemia residing in the neonatal intensive

g e n e t i c a r r a n g e m e n t s in b a c t e r i a l c h r o m o s o m e
(9, 10). T h e y a r e s u f f i c i e n t l y h e t e r o g e n o u s to
make suitable targets for comparison of
different bacterial species (10-15). A single
Escherichia coli 16+23S r R N A p r o b e c a n b e
efficiently applied for differentiation of
d i f f e r e n t s p e c i e s of g r a m - n e g a t i v e b a c i l l i (10, 16)
d u e to h i g h l y c o n s e r v e d n u c l e o t i d e s e q u e n c e s
w i t h i n c e r t a i n r e g i o n s o f r R N A genes.
In t h e p r e s e n t s t u d y , we d e v e l o p e d a m e t h o d for
ribotyping of coagulase-negative staphylococci
r e p r e s e n t e d b y 15 d i f f e r e n t s p e c i e s a n d c o m -
p a r e d this m e t h o d w i t h c h r o m o s o m e typing. I n
addition, we conducted a preliminary evaluation
of the method by differentiation of coagulase-
negative staphylococci isolated from our
n e o n a t a l i n t e n s i v e c a r e unit.
care unit were examined. The reference strains are listed in
Table 1. The clinical strains consisted of the following
species: Staphylococcus epidermidis (n = 29), Staphylo-
coccus haemolyticus (n = 19), Staphylococcus warneri
(n = 3) and Staphylococcus capitis (n = 2).
DNA Isolation, Restriction Endonuclease Digestion and
Fractionation by Agarose Get Electrophoresis, DNA was
purified by a cleared lysate procedure as previously
described (4). Total DNA was digested in 6--10 I.tg amounts
at a concentration of 200 I,tg/ml with 5-8 unit of restriction
enzyme (Pharmacia Fine Chemicals, USA) per l g g of
DNA for 5 h, under conditions recommended by the
supplier. Each digest was repeated at least twice to ensure
that gels represented complete and not partial digest.
Agarose gel electrophoresis was carried in horizontal slab
gels of 0.6 % agarose (IBI, USA) in TBE buffer (0.089M
Tris-borate, 0.089M boric acid and 0.002M EDTA) at room
temperature at 2.5 V/cm for 20 h. The gels were stained with
ethidium bromide (1 gg/ml) and photographed under
ultraviolet illumination (Fotodyne UV300) with a Wratten
2A filter using Polaroid type 55/PN film.

End-Labeling of rRNA. Commercially available 16+23S

Materials and Methods
Bacterial Strains. A total of 25 reference strains from the
American Type Culture Collection and 53 clinical strains
isolated from blood and mucosal surfaces of 19 premature
rRNA from Escherichia colt (Pharmacia Fine Chemicals,
catalogue no. 27-2512-01) was used. The rRNA was
dephosphorylated with calf-intestinal phosphatase
(Pharmacia Fine Chemicals) extracted once with
phenol/chloroform (Iv:Iv), four times with ether and
ethanol precipitated. The rRNA was labeled with [7-32p]-

Table 1: Results of chromosome typing and ribotyping of eoagulase-negative staphylococcal

reference strains from the American Type Culture Collection.

Lane no. Organism ATCC Chromosome

in Figure 1 no. typea Rib°typ eb
1 Staphylococcus auricularis 33753 1.1 a
2 Staphylococcus capitis 35661 2.2 b
3 Staphylococcus capitis 27841 2.2 c
4 Staphylococcus caprae 35538 3.1 d
5 Staphylococcus cohnii 29972 4.2 e
6 Staphylococcus cohnii 29973 4.2 e
7 Staphylococcus epidermidis 35983 5.3 f
8 Staphylococcus epidermidis 35984 5.3 g
9 Staphylococcus gallinarum 35539 6.1 h
10 Staphylococcus haemolyticus 29968 7,3 i
11 Staphylococcus haemolyticus 29969 7.I j
12 Staphylococcus haernoIyticus 29970 7.1 )
13 Staphylococcus horninis 27844 8.3 k
14 Staphylococcus hominis 27845 8.3 1
15 Staphylococcus hominis 27846 8.3 m
16 Staphylococcus intermedius 29663 9.1 n
17 Staphylococcus lentus 29070 10,1 o
18 Staphylococcus saprophyticus 15305 11.3 p
19 Staphylococcus saprophyticus 35552 11.3 r
20 Staphylococcus sciuri 29062 t2.1 s
21 Staphylococcus simulans 27851 13.1 t
22 Staphylococcus warneri 27836 14.3 u
23 Staphylococcus warneri 27837 14.3 v
24 Staphylococcus warneri 27839 14.3 u
25 Staphylococcus xylosus 35663 15.1 w

aTwenty-four chromosome types designated 1.1 to 15.1 are presented in Figure 1A. Strain-specific
chromosome types are designated 0.1 for identical strains displaying _>95 % similarity (% S) and 0.2
to 0.3 for different strains displaying respectively from 85-95 % to 60 < % S < 85. % S values were
determined as described in Materials and Methods.
bTwcnty-two ribotypes a to w are presented in Figure lB. Identical ribotypes had _>95 % S.
590
ATP by using T4 polynucleotide kinase (Promega, USA)
as previously described (17). The specific activity of the
probe was at least 5 x 108 cpm/gg of RNA.
Ribotyping. rDNA patterns were obtained by restricting
total DNAs with restriction endonuclease, electro-
phoresing the fragments on agarose gels, transferring the
fragments to nitrocellulose membranes (Schleicher &
Schuell, USA) as described by Southern (18) and
hybridizing them with end-labeled 16+23S rRNA from
Escherichia coil The prehybridization and hybridization
solution contained 50 % formamide, 4 x SSC (1 x SSC
= 0.15M Na chloride, 0.015M Na citrate pH 7.0), 0.5 %
sodium dodecyl sulfate (SDS), 0.02 % bovine serum
albumin fraction V, 0.02 % Ficol, 0.02 % polyvinyl
pyrrolidone and lmM EDTA. For each blot, 105 cpm of
rRNA probe was added to I ml of hybridization solution.
The filters were generally prehybridized for a minimum of
2 h before addition of the probe. After 12-18 h
hybridization at 42 °C the filters were washed once in
2 x SSC, 0.1% SDS at room temperature for 10.min,
followed by two 20 rain washes in 0.2 x SSC, 0.1% SDS at
75 °C and one 20 min wash in 0.1 x SSC, 0.1% SDS at 50 °C.
The filters were then exposed to Kodak XAR-Z film at
- 70 °C with an intensifying screen.
Numerical Analysis of DNA Patterns. Electrophoreto-
grams of restriction endonuclease fingerprinting (REF)
patterns of chromosomal DNA on negative film and
rDNA patterns on X-ray film were scanned with a laser
densitometer (Ultroscan XL model 2222, Pharmacia LKB
Bioteehnology, Sweden). The absorbance values were
directly transferred to an IBM PC/AT compatible
computer using a serial connection. The number of data
points recorded were 1875 per 75 ram-long scan for REF
and 363 per 135 ram-long scan for rDNA patterns. The
densitometry traces from different gels were aligned and
the start and the end of the trace were respectively defined
between a 2 to 35 kilobase pairs region for REF and 1 to 25
kilobase pairs region for rDNA patterns. The number of
data points in each trace was reduced to a standard 300-
point trace and 100-point trace for REF and rDNA
patterns, respectively, using an interpolation program
(TPROCESS). The background cutoff was set at 1.0.
Similarities between compared pairs of DNA patterns
were assessed using the correlation coefficient of Sneath
and Sokal (19) incorporated intoTMATRIX. Cluster
analysis was performed using TCLUSTER, TPROCESS
and TMATRIX which were originally written by Jackman
et al. (20) in Commodore PET BASIC and adapted in
Microsoft QuickBASIC to run on IBM/AT compatible
computers.
DNA Fragment Size Determination. The size of rDNA
fragments hybridizing with labeled rRNA of Escherichia
coli was calculated from electrophoretic migration by
comparisons with 32p-labeled HindlII fragments of lambda
DNA (23.1, 9.4, 6.6, 4.3, 2.3, 1.9 kilobase pairs) and 32p_
labeled linear ladder markers (Bethesda Research
Laboratory, USA). The method of multiple regression of
log10 molecular size against lOgl0 relative mobility and the
reciprocal square root of the relative mobility was used for
estimating sizes of DNA fragments (21).
Results
Analysis of Reference Strains. To assess the dis-
criminating p o w e r of ribotyping, total D N A
f r o m 25 r e f e r e n c e strains was digested with a
Eur. J. Clin. Microbiol. Infect Dis.
variety of restriction enzymes and analyzed by
S o u t h e r n b l o t h y b r i d i z a t i o n . O f the eight
restriction e n d o n u c l e a s e s that were e x a m i n e d
(AvaI, BamHI, ClaI, EcoRI, HaeIII, PstI, PvuII
and SacI) ClaI provided the best discrimination
a m o n g d i f f e r e n t s p e c i e s a n d s t r a i n s of
coagulase-negative staphylococci.
T w e n t y - t w o distinct strain-specific ribotyp.es
w e r e p r o d u c e d with ClaI a m o n g the 25 strams
a n a l y z e d ( T a b l e 1). T h e s e r i b o t y p e s w e r e
r e p r e s e n t e d by patterns which consisted of five
to ten h y b r i d i z a t i o n b a n d s c o r r e s p o n d i n g to
D N A fragments encoding r R N A ( r D N A ) in the
m o l e c u l a r r a n g e of 1.4 to 24.2 kilobase pairs
(Figure 113). T h e degree of sequence h o m o l o g y
b e t w e e n the Escherichia coli 16+23S r R N A
p r o b e and the corresponding regions of r R N A
genes of coagulase-negative staphylococci was
reflected by different intensities of hybridiza-
tion bands. T h e ratio of high-intensity to tow-
intensity hybridization bands was stable u n d e r
the s a m e m o d e r a t e l y stringent hybridization
conditions used in this study. The stability of
r i b o t y p e s . w a s e x a m i n e d by analysis of initial
and final patterns of hybridization bands of all
25 reference strains after three in vitro passages
on agar. T h e p a t t e r n of each strain e x a m i n e d
remained unchanged.
W h e n c o m p a r i n g the two m o l e c u l a r typing
procedures, c h r o m o s o m e typing and ribotyping,
it was o b s e r v e d that the c h r o m o s o m e typing
p r o c e d u r e distinguished 24 r e f e r e n c e strains
w h e r e a s r i b o t y p i n g distinguished 22 strains
(Figure 1, T a b l e 1).
Analysis of Clinical Isolates. The ribotypes were
c o m p a r e d with c h r o m o s o m e types a m o n g 53
clinical isolates of coagulase-negative staphylo-
cocci f r o m the neonatal intensive care unit. T h e
following n u m b e r s of different r i b o t y p e s and
c h r o m o s o m e t y p e s p r o d u c e d by ClaI w e r e
observed respectively: three and four f r o m 29
strains of Staphylococcus epidermidis, two and
two f r o m 19 s t r a i n s of S t a p h y l o c o c c u s
haemolyticus, one and three f r o m three strains
of Staphylococcus warneri, one and two f r o m
two strains of Staphylococcus capitis. The two
strains of Staphylococcus warneri which were
not discriminated by ribotyping with ClaI were
distinguished using HpaI (Figure 2, lanes 6, 7).
T h e f r e q u e n c y of distribution of distinct ribo-
types was analyzed a m o n g isolates of coagulase-
negative staphylococci obtained from blood and
mucosal sites. All three ribotypes f, f.1 and g.2
were o b s e r v e d a m o n g b o t h blood and mucosal
isolates of Staphylococcus epidermidis re-
covered f r o m eight infants. T w o pairs of distinct
ribotypes f and L1, and f and g.2 were isolated
f r o m four infants. In contrast, only one j.1 of the
two r i b o t y p e s j and j.1 of S t a p h y l o c o c c u s
 Vol. 9, 1990 591

A 1 2 3 4 S 6 ? 8 9 10 11 IR lS 14 18 16 17 18 19 :~0 21 22 23 24 26

B I 2 s 4 s • y a*lo:llisla~41SleiVwn2ollllBNx

23.2
- * - - - - - 12.0

q 6.2

4 4.0 Agarose gel electrophoresis of

F i g u r e 1:
total DNA after digestion with ClaI (A).
Autoradiogram of Southern blot of ClaI-
,.ll digested DNA after hybridization with 32p_
labeled rRNA from Escherichia coli (B).
~- 2.0 Lanes are as described in Table 1.

emilm

haemolyticus appeared in four blood culture

Clal Hpal isolates while both types appeared among 15
1 2 3 4 5 6 ? 1 2 3 4 5 6 ?
mucosal isolates of the same organism (data not
23.2 shown). In a few cases examined, ribotypes
identified from blood isolates were similar to
12.0
those of the corresponding mucosal isolates.
When mucosal and blood isolates were analysed
6.2 in eight and three bacteremic neonates infected
with Stapyhlococcus epidermidis and Staphylo-
4.0 coccus haemolyticus respectively, six of eight
blood isolates of Staphylococcus epidermidis
and all four isolates of S t a p h y l o c o c c u s
haemolyticus had similar ribotypes to those of
mucosal isolates (data not shown).
2.0

Comparison of Strain-Specific Ribotypes among

Six Coagulase-Negative Staphylococcus Species.
~g~o.qllJWlJ Comparative analysis of ribotypes within the
species Staphylococcus capitis, Staphylococcus
epidermidis, Staphylococcus haemolyticus,
Figure 2. Ribotyping performed with HpaI was more Staphylococcus hominis, Staphylococcus sapro-
discriminating than with ClaI for some clinical isolates of phyticus and Staphylococcus warneri, each
coagulase-negative staphylococci. Lanes are described by represented by a minimum of two reference
ribotypes produced with ClaI.Lanes 1 to 3: Staphylococcus
epidermidis ribotypes f, g.2 and f.1. Lanes 4 to 5: strains, d e m o n s t r a t e d the presence of strain-
Staphylococcus haemolyticusribotypes j and j.1. Lanes 6 to specific ribotypes (Figures 3). These ribotypes,
7: Staphylococcus warneriribotype u. represented by patterns of hybridization bands,
592 Eur. J. C/in. Microbiol. Infect Dis.

= I
d d
t t I t
libHlCt b c f g K 2 f l i j j l k | m p r

Figure 3: Schematic representation of the

strain-specific ribotypes obtained after
digestion of total DNA with ClaI and
hybridization with 32p-labeled rRNA from
Escherichia coli. Ribotypes b-v represent
reference strains described in Table 1. Ribo-
types g.2 and f.1,Staphylococcus epidermidis
m
clinical isolate no. 380 and no. 207; ribotype
-- ~ '-~ 6.2 j.1, Staphylococcus haemotyticus clinical
m
isolate no. 255. Additional digits denote the
m
w
differencein the presence or migrationof 1-
2 bands. A thick line indicates a band that is
m m -- 4.0 found in most strains of a given species. A
ribotype represented by a pattern consisting
m of only thick bands is denoted here as a
species-specific i'core" ribotype. Sizes of
u m rDNA fragments were calculated by the
method described in Materials and
Methods. Numbers to the right represent
2.0
32p-labeled linear DNA size markers (in
kilobase pairs).
m

differed from one another by the number and staphylococcal species and individual strains
size of rDNA restriction fragments. Most of within species based upon restriction site
those patterns within the same species were heterogeneity within their 16S and 23S rRNA
highly related and displayed from 65 to 100 % operons. This approach was originally intro-
common bands and greater than 65 % similarity duced by Grimmont and Grimmont (10) and has
values. Patterns consisting of common bands been used to study the epidemiology of a variety
present in the majority of strains of a given of bacterial infections (11-15). Recently,
species are described in this study as re- De Buyser et al. (22) have applied 16S rDNA
presenting species-specific "core" ribotypes from Bacillus subtilis as a probe to compare
(Figure 3). There were, however, a few in- rRNA gene restriction patterns of Staphylo-
dividual reference strains within the species coccus spp. Other investigators have demon-
Staphylococcus hominis and Staphylococcus strated the usefulness of selected fragments of
h a e m o l y t i c u s that shared less than 50 % bacterial chromosome of known (5, 6, 7) or
common bands with the rest of strains of the unknown (8) function to highlight restriction
same species. Staphylococcus haernolyticus site heterogeneity in the corresponding regions
ATCC 29968 and ATCC 29969, represented by of bacterial chromosome. Analysis of strains
distinct eight-band patterns (Figure 3, ribotypes representing 15 different species of coagulase-
i and j), had only four common bands. negative staphylococci revealed simple patterns
Staphylococcus hominis ATCC 27844 and of five to ten hybridization bands produced after
ATCC 27846, characterized by distinct six- and digestion with ClaI. Hence, it is estimated that
ten-band patterns (Figure 3, ribotypes k and m), coagulase-negative staphylococci contain at
displayed only two common bands. least five copies of 16S and 23S rRNA operons.
This study demonstrated that ribotyping which
combines Southern hybridization of chromoso-
mal DNA fingerprints with the use of the
Discussion Escherichia coti rRNA probe discriminates
between various species and individual strains
The present study assessed the application of the of coagulase-negative staphylococci. One to
16+23S rRNA of Escherichia coli as a probe for several strain-specific ribotypes were observed,
the d i f f e r e n t i a t i o n of coagulase-negative depending on the degree of divergence within a
 VoI. 9, i990
given species. This indicated strain-to-strain
variation in certain regions of otherwise highly
conserved rRNA genes. Most hybridization
patterns representing strain-specific ribotypes
were highly related and displayed from 65 to
100 % common bands. Patterns consisting of
bands present in the majority of examined
strains within the same species were identified
in this study and described as representing
species-specific "core" ribotypes (Figure3).
Additional studies are required to verify further
the description of the species-specific ribotypes.
A marked heterogeneity of certain strain-
specific ribotypes was observed in Staphylo-
coccus warneri, Staphylococcus haemolyticus
and Staphylococcus hominis species. This
finding is in agreement with our earlier
observation on the heterogeneity of patterns of
chromosomal DNA restriction fragments within
the same coagulase-negative species (manuscript
in press). We speculate that a variety of specific
environmental habitats and the abundance of
extrachromosomal elements, such as plasmids
and temperate bacteriophages, can contribute to
the polymorphism of the members of coagulase-
negative staphylococcal species. The importance
of examination of genetic arrangements of
chromosomal genes and their flanking regioas,
rather than nucleotide sequences of those genes
for identification of bacterial species and
subspecies, has been previously indicated by
Riley and Anilionis (23). It remains to be
established, however, whether ribotyping could
be used as a supplementary system to DNA-
DNA hybridization studies or whether it could
be used separately to contribute specific
information on taxonomy of coagulase-negative
staphylococci.
Ribotyping appeared to be slightly less dis-
criminating than chromosome typing. Classifi-
cation of Staphylococcus haemolyticus isolates
was identical by both methods. Ribotyping
distinguished three and chromosome typing
distinguished four major groups among the 29
Staphylococcus epidermidis isolates. It is of
interest that Staphylococcus epidermidis strains
representing two related ribotypes f and f.1
belonged to three related major chromosome
types which displayed similarity values from 80
to 95 % (data not shown). Specificity of ribo-
typing was improved for typing of Staphy-
lococcus warneri when restriction enzymes
HpaI or AvaI were employed. This finding
confirms observations of others regarding the
significance of the use of a variety of restriction
enzymes in the ribotyping procedure (11, 16, 24).
A major advantage of using ribotyping for
species and strain discrimination for epi-
demiologic studies is that it provides simple
patterns that can be easily subjected to multiple
593
comparisons with those previously recorded.
The expected requirement for such comparison
would be the use of similar conditions of
agarose gel electrophoresis, the use of the same
fragment size markers and the same formula for
size calculations.
The epidemiological application of ribotyping
to examination of coagulase-negative staphylo-
coccal infections was not thoroughly evaluated
in this study. Our preliminary results suggest
that the method may be of epidemiological value
in tracing the source and mode of distribution of
coagulase-negative staphylococci strains in a
neonatal intensive care unit. To assess further
the usefulness of ribotyping versus chromosome
typing for studying coagulase-negative staphy-
lococcal infections, a prospective examination
of strains colonizing and infecting neonates
admitted to a neonatal intensive care unit using
both molecular typing techniques will be
conducted. Whether specific ribotypes and/or
chromosome types are associated with higher
pathogenicity of blood strains remains to be
established.
Acknowledgements
This investigation was supported in part by the Banting
Foundation.
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