Genomic fingerprinting of penicillinase-producing strains of Neisseria gonorrhoeae in Valencia, Spain.
ABSTRACT To compare the value of different markers and their combinations with the restriction enzyme technique in the differentiations of penicillinase-producing N. gonorrhoeae (PPNG) strains.
17 PPNG strains isolated from symptomatic, untreated male patients with urethritis were characterised by antibiotic sensitivity testing, auxotyping, serotyping, plasmid profile, and restriction endonuclease fingerprinting (Hind III digestion). Cluster analysis with the method of unweighted pair-group average (UPGMA) linkage was used to calculate similarity or dissimilarity for PPNG strains.
Either auxotyping or plasmid profile alone differentiated three groups of PPNG strains, whereas the combination auxotyping/serotyping identified 10. Although the combination auxotyping/serotyping/plasmid profile and the restriction enzyme technique showed a similar discrimination ability (differentiation of 11 PPNG strains), genomic fingerprinting gave highly specific restriction patterns on individual gonococcal isolates.
The combination of different markers gave more epidemiological information than the use of only one. The sequence of discriminating ability for PPNG strains was: auxotyping/serotyping less than auxotyping/serotyping/plasmid profile less than restriction patterns of genomic DNA.
- Acta pathologica et microbiologica Scandinavica. Section B: Microbiology and immunology 02/1971; 217:Suppl 217:1+.
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ABSTRACT: Twenty gonococcal isolates representing different serovars of the protein IA & IB serogroups, as determined by co-agglutination with monoclonal antibodies, were investigated by the restriction enzyme (RE) technique with Hind III enzyme. The patterns resulting from RE digestion and subsequent electrophoresis of the DNA fragments in gel consisted of 40–45 bands. Eight RE patterns were observed among ten of the protein IA strains representing three serovars with an identical pattern for three strains of the same serovar with positive epidemiology. Seven RE patterns were observed among ten of the protein IB strains representing five serovars with identical patterns for each of three pairs with available positive epidemiology for two of them. Seven samples from the same bacterial clone extracted and digested separately showed identical band patterns. The genetic stability was illustrated by identical patterns through 41 passages in vitro and by the same band pattern of the various virulent and avirulent colony morphology variants of a particular gonococcal strain. The results show that RE analysis of the gonococcal DNA genome is well suited as an adjunct to phenotypic markers in epidemiological studies of gonorrhoea.Apmis 08/1984; 92B(1‐6):271 - 278. · 2.07 Impact Factor
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ABSTRACT: A strain of Escherichia coli is described that contains eight plasmid species ranging in size from 1.36 × 106 to 35.8 × 106 daltons. This strain can be employed as a single source of covalently closed circular deoxyribonucleic acid molecules of different sizes for use as references in agarose gel electrophoretic analysis.Plasmid 07/1978; 1(3):417-20. · 1.28 Impact Factor
Genitourin Med 1992;68:170-173
Genomic fingerprinting of
penicillinase-producing strains of Neisseria
gonorrhoeae in Valencia, Spain
M A Dasi, J M Nogueira, J J Camarena, C Gil, R Garcia-Verdu', J L Barbera, J Barbera
Objective-To compare the value of dif-
ferent markers and their combinations
with the restriction enzyme technique in
the differentiations of penicillinase-pro-
ducing N. gonorrhoeae (PPNG) strains.
Materials and methods-17 PPGN strains
isolated from symptomatic,
male patients with urethritis were charac-
ternsed by antibiotic sensitivity testing,
auxotyping, serotyping, plasmid profile,
and restriction endonuclease fingerprint-
ing (Hind III digestion). Cluster analysis
with the method ofunweighted pair-group
average (UPGMA) linkage was used to
calculate similarity or dissimilarity for
plasmid profile alone differentiated three
groups of PPNG strains, whereas the
combination auxotypinglserotyping iden-
tified 10. Although the combination aux-
otyinglserotypinglplasmid profile and the
restriction enzyme technique showed a
similar discrimination ability (differen-
tiation of 11 PPNG strains), genomic
fingerprinting gave highly specific restric-
tion patterns on individual gonoccocal
Conclusions-The combination of differ-
ent markers gave more epidemiological
information than the use of only one. The
sequence of discriminating
PPNG strains was: auxotypinglserotyping
< auxotypinglserotypinglplasmid profile <
restriction patterns of genomic DNA.
M A Dasi
J M Nogueira
J J Camarena
J L Barbera
Address for correspondence:
J M Nogueira, MD,
Facultad de Medicina, Avda.
Blasco Ibaiiez 17, 46010
Accepted for publication
20 February 1992
The incidence of penicillinase
mase)-producing strains of Neisseria gonor-
rhoeae has been steadily increasing in most
European countries, including Spain.' Pheno-
typic and genotypic characterisation of these
strains has proved valuable for epidemiological
tracing in gonococcal disease. Auxotyping,2
antibiotic susceptibility testing,3
ing,4-9 plasmid analysis, and the so-called
genomic fingerprinting based on restriction
entiating between penicillinase-producing N.
strains. Genomic fingerprinting
gives highly reproducible, stable, and specific
restriction patterns on individual gonococcal
isolates'2``6 and can serve as a useful adjunct
to serological classification.
of the gonococcal
`have been used by different research
as epidemiological tools for differ-
We compared the value of different markers
and their combinations with the restriction
enzyme technique in the differentiation of
penicillinase-producing strains of N. gonor-
Materials and methods
A total of 17 penicillase-producing N. gonor-
untreated male patients with
Valencia, Spain, between 1985 and 1988 were
included in the study. They accounted for
9.9% of all isolates of N. gonorrhoeae during
All isolates were confirmed as N. gonorrhoeae
by colony morphology, Gram stain, oxidase
reaction, and acid reproduction from glucose
but not maltose, sucrose, lactose or fructose.
Purified stock cultures were maintained in
trypticase-soy broth (BBL Microbiology Sys-
tems, Cockeysville, MD, USA) containing
20% glycerol and stored at - 70°C. Isolates
were subcultured on GC agar medium base
(Oxoid, UK) with 1%
(BBL), 1% (v/v) IsoVitalex (BBL) and without
antibiotics, and incubated at 37°C with 5%
CO2 atmosphere in a humidified incubator for
20 h. The strain number was that assigned in
The isolates were tested for fi-lactamase
production by means of the chromogenic
Antibiotic sensitivity testing
We tested sensitivity to antibiotic by an agar
dilution method3 using GC agar base enriched
with 1% (v/v) IsoVitalex containing various
concentrations (0.125 to 128 mg/l) of pen-
icillin G; 104 colony forming units (cfu) were
transferred to the agar surface. The minimum
inhibitory concentration (MIC) was defined as
the lowest concentration that completely sup-
pressed bacterial growth after 18-24 h incu-
Auxotyping was performed by growing N.
gonorrhoeae on chemically defined medium as
described previously.2 Strains were tested for
their requirements for proline (Pro-), arginine
(Hyx -), and uracil (Ura ) or combinations of
these requirements. Strains with no special
requirements regarding these substances were
called prototrophic (Proto).
Penicillinase-producing gonococcal strains
ing minimum inhibitory concentrations (MIC), plasmid size, auxotype, and serogroup
and serivar patterns.
Distribution of 17 penicillinase-producing Neisseria gonorrhoeae strains regard-
4-5 and 24-5
Plasmids were isolated by a rapid alkaline
extraction procedure as described by Birnboim
and Doly,'8 with some modifications.'9 Plas-
mids were analysed in 0-8% agarose gels. The
Escherichia coliV517 strain20 (size estimates of
plasmids 1.8, 2.0, 2.6, 3.4, 3.7, 4-8 and 35-8
106 daltons) and N. gonorrhoeae strains CCUG
5449 (size estimates of plasmids 3.2 and 2 6
106 daltons) and CCUG 6016 (size estimates
of plasmids 24-5, 4-5 and 2-6 106 daltons)
(Cuture collection, University of Goteborg,
Sweden) with plasmids of known molecular
weight were included as fragment size mark-
Hospital, Stockholm, Sweden). Each strain
was tested against a set of five antibodies
specific to protein AI (designated Ar, Ao, As,
At, and Av) and nine antibodies specific to
protein IB (Br, Bo, Bp, By, Bv, Bu, Bs, Bt, and
Penicillinase-producing strains of N. gonor-
rhoeae were distributed into serovars according
to their reactions with the different monoclonal
antibody reagents.The capital letters A (forWI
strains) and B (for WII/WIIII) were followed
by lower case
letters representing positive
reactions with the corresponding coagglutina-
Restriction endonuclease fingerprinting
The restriction enzyme technique was per-
formed as described by Falk et all' with some
modifications. The restriction endonuclease
used was Hind III (Boehringer GmbH, Mann-
heim, Germany). After electrophoresis, gels
were stained with silver (Bio-Rad Lab., Rich-
mond, CA, USA) or alternatively with ethi-
scanned with an LKB2202 UltroScan laser
densitometer (Pharmacia LKB, Uppsala, Swe-
den) provided with a Hewlett-Packard 3390
integrator. The molecular weight ofeach diges-
ted DNA fragment was calculated by a com-
puter program of robust estimation described
by Plikaitis et al.22
Statistical analyses of data for grouping differ-
ent isolates were carried out by assigning to
each electrophoretogram a dimensional vector
absence =0), where "n" was the total number
of resulting DNA fragments. These vectors
were introduced into a program of cluster
analysis using the "proximities" procedure23
according to the following equation: SM (x, y)
= a + d/a + b + c + d, where SM (x, y) was the
similarity coefficient between two strains; "a"
_the number of DNA fragments which were
present in both strains; "b" the number of
DNA fragments which were present in the "x"
strain and absent in the "y" strain; "c" the
number ofDNA fragments which were present
in the ''y'' strain and absent in the uxni
and "d" the number ofDNA fragments which
were absent in both strains. Once this matrix
been calculated, the cluster analysis with
the method of unweighted pair-group average
(UPGMA) linkage was used.
obtain a graphic representation (dendrogram),
the program used converted the similarity
(rescaled distance cluster combine) according
~~~~~~~to the following formula:
Serological classification into serogroups (WI,
WII, andWIII) and serovars was performed by
using a set of monocolonal antibodies bound
to staphylococci containing protein A (provi-
ded by S Bygdeman, Huddings University
RD (x, y) =x
SMmax -SM min
where, "Smax" and "Smin" were the values of
the maximum and minimum similarity coeffi-
cients and "N" the index of the scale. Analyses
Honeywell Bull computer.
tetic heterogeneity ofN. gonorrhoeae strains after DNA digestion with Hind
imbers 1-8 corresponding to strains 52, 171, 102, 93, 185, 20, 186,and
III (lane nu
103). bp, b
Dasi, Nogueira, Camarena, Gil, Garcia-Verdu, Barbera, Barberd
All isolates were fl-lactamase producers with
MIC values of penicillin ranging between 8
and >128 mg/l (table). Three different auxo-
types were identified. Nine of the 17 strains
were Proto, seven were Pro
The 3.2megadaltonor Africa(Af-)plasmid
was found in 12 (70.6%) strains. Four strains
contained the4-5 megadalton or Asia(As-)
plasmid. In one strain the conjugative 24-5
megadalton plasmid coexisted with the 4-5
megadalton plasmid (As'). The cryptic 2-6
megadalton plasmid was present in
SerogroupWI wasrepresented bytwo iso-
lates which belonged to the serovars Arst and
Av. SerogroupWII/WIII was represented by 1 5
(table).The serovarpattern Bropwas identi-
fiedin 35% of the isolates.
Restriction patterns obtained with Hind III
digestion ofgenomic DNA of different isolates
consisted of a mean of 50 bandsper patternof
which size offragmentsbetween2-4and1-3
kilobase pairs (containing a median of 25 to
30 bands) were studied (fig 1). Three common
bands with fragment sizes of 2-1, 1-5, and 1-3
kilobase pairs, respectively, were found in all
lanes. Highly resolved bands standing out
minimal differences between two isolates were
obtained with silver stain (fig 2).
Statistical grouping of isolates showed simi-
larity coefficients between 1.0 and 0-7456 [RD
(x, y) 0 and 17, respectively]. With a cutoff
level at rescaled distance cluster [RD (x, y)] of
8, four clearly defined clusters of N. gonor-
rhoeae strains were identified. Figure 3 shows
the grouping dendrogram.
Discrimination by different markers and
their combinations is shown in fig 4. Auxotyp-
ing differentiated three groups of strains of N.
gonorrhoeae, whereas the combination auxotyp-
five prototrophic IB/rop strains, auxotyping/
serotyping did not discriminate four strains
with the 4-5 megadalton plasmid(As
one strain with the 3-2 megadalton plasmid
(AfM ). When the combination auxotyping/
serotyping/plasmid profile was used, a distribu-
tion of N. gonorrhoeae strains into 11 groups
was obtained. The restriction enzyme pattern
, and only one was
| : 0e
(lanes 1 and 2,
) with identical
egard to 2 or 4
10. Among the
Rescaled distance cluster combine
Clusters of strains (a, b, c,
j) at the 8 cutoff level.
Ar Al Arstg
penicillinase-producing N. gonorrhoeae strains.
Auxotypes: Proto (prototrophic), Pro
(arginine). Plasmid profile and groups obtained with
DNA restriction patterns, *3.2 megadalton plasmid
(Af ), **4.5 megadalton plasmid (As
megadalton plasmid (As').
Phenotypic and genotypic discrimination of 17
), t24-5 and 4 5
although it detected differences between two
Af- strains (isolates 185 and 167) which were
completely identical by combining the other
markers. However, the restriction enzyme pat-
tern was unable to differentiate isolates 171
and 186 (fig 4).
The present study was undertaken to compare
the value of different markers as epidemio-
logical tools in the characterisation of peni-
cillinase-producing strains of N. gonorrhoeae in
our geographical area.
In this particular material, both auxotyping
and plasmid profiles were of limited value. We
found a dominance of the African type strains
carrying a 3.2 megadaltonf-lactamase encod-
others.'14Serological classification with spe-
cific monoclonal antibodies enhanced the abil-
strains. Auxotyping combined with serotyping
gave more epidemiological information than
the use of one of these only. In five proto-
trophic IB/rop strains, auxotyping/serotyping
did not differentiate the African from the Asian
type strains, but plasmid profiles as a comple-
ment to both markers permitted such a dif-
The distribution of gonococcal strains into
1 1 groups obtained with the combination of
to discriminate between closely related
99 and 64
only with r
Penicillinase-producing gonococcal strains
three markers (auxotyping/serotyping/plasmid
profiles) was in agreement with the genomic
fingerprinting. The restriction enzyme tech-
nique may differentiate between strains that by
phenotypic classification appear to be identi-
cal. The reproducibility and reliability of the
method has been demonstrated by other inves-
tigators.13-1625 Moreover, the use of silver stain
has been proved
The characterisation of a clone as As' IB/rop
strains may have been linked to an outbreak
that occurred in our urban area at the end of
1987. The two Af- IB/x strains in which all
markers and their combinations coincided,
were similar to those described in an outbreak
of penicillinase-producing strains of N. gonor-
rhoeae in the
city of Barcelona
Valencia) in 1987.26
The combination of different markers has
information than the use ofonly one. Although
the restriction enzyme technique appears to be
highly specific, it is both laborious and expen-
sive and usually limited to special laboratories.
We conclude that the combination of sero-
logical classification with specific monoclonal
antibodies with auxotyping and/or plasmid
profiles is of great potential use for epidemio-
logical and clinical studies. Genotypic finger-
printing could be of practical importance for
detecting subtle genetic differences of pheno-
typically similar gonococcal clones.
a valuable alternative
This work was supported in part by "Fondo de Investigaciones
Santarias de la Seguridad Social" (FIss) grant 90/0613. We
thank Marta Pulido, M.D. for editorial assistance and copy
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