712
Carriage of Antibiotic-Resistant Streptococcus pneumoniae by Children in
Eastern and Central Europe-A Multicenter Study with Use of Standardized
Methods
P.
B.
L.
P.
C. Appelbaum, C. Gladkova, W. Hryniewicz,
Kojouharov, D. Kotulova, F. Mihalcu, J. Schindler,
Setchanova, N. Semina, J. Trupl, S. Tyski,
Urbaskova, and M. R. Jacobs
From the Hershey Medical Center, Hershey, Pennsylvania, United
States; Central Institute for Epidemiology, Moscow, Russian Federation;
Medical Academy, Sofia, Bulgaria; Sera and Vaccines Central Research
Laboratory, Warsaw, Poland; Institute Cantacuzino, Bucharest,
Romania; National Reference Laboratory for Antibiotics, Prague, Czech
Republic; National Cancer Institute and Institute of Microbiology,
Bratislava, Slovak Republic; and Case Western Reserve University,
Cleveland, Ohio, United States
With use of standardized techniques, a study of nasopharyngeal pneumococcal carriage in children
in six Central and Eastern European cities was undertaken during the winter of 1993-1994. Nasopharyngeal swab specimens were collected from 954 children (predominantly under the age of 5
years) who were hospitalized or attending outpatient clinics or day-care centers. Susceptibility of
isolates was determined by disk diffusion (on Mueller-Hinton agar with 5% sheep blood). Disks
containing 1 p,g of oxacillin were used to screen for susceptibility to penicillin G. Pneumococci were
recovered from 258 (27.0%) of the 954 children. A variety of strains were recovered, and most
penicillin-resistant strains were resistant to multiple agents. Minimum inhibitory concentrations of
penicillin for selected resistant strains were 0.125-8 p,g1mL. Resistance to penicillin was common
in strains from Bulgaria, Romania, and Slovakia. Resistance to erythromycin and chloramphenicol
occurred in Bulgarian and Romanian strains. Strains from Poland were all susceptible to penicillin,
but many were resistant to tetracycline. Resistance to trimethoprim-sulfamethoxazole was common
in Bulgarian, Romanian, and Slovak strains. Czech and Russian strains were predominantly susceptible to antibiotics. Most resistant strains were of serotypes 6, 14, 19, and 23.
Infections caused by Streptococcus pneumoniae are a leading
cause of illness and death among young children, individuals
with debilitating medical conditions, and the elderly. S. pneumoniae is the most commonly identified bacterial cause of otitis
media and pneumonia in most parts of the world. Treatment
of most infections remains empirical because of lack of access
to suitable diagnostic specimens in cases of pneumonia, sinusitis, and otitis media as well as difficulty in interpretation of
sputum cultures [1- 3].
Current treatment regimens are based mainly on historical
data and findings in studies of patients infected with fully
susceptible strains [2]. However, the emergence of strains of
S. pneumoniae resistant to penicillin - and often resistant to
other classes of antimicrobial agents as well-complicates empirical management of meningitis and otitis media. In addition,
Received 23 October 1995; revised 18 April 1996.
Grant support: Supported in part by F. Hoffmann-LaRoche, Ltd., Basel,
Switzerland, and Marion Merrell Dow (Europe), Horgen, Switzerland.
Informed consent was obtained from patients or their parents/guardians, and
guidelines for human experimentation of the U.S. Department of Health and
Human Services and of the authors' institutions were followed in the conduct
of the study.
Reprints or correspondence: Dr. Peter C. Appelbaum, Department of Pathology, Hershey Medical Center, P.O. Box 850, Hershey, Pennsylvania 17033.
Clinical Infectious Diseases 1996; 23:712-7
© 1996 by The University of Chicago. All rights reserved.
1058-4838/96/2304-0006$02.00
optimal management of patients with infections caused by resistant pneumococci remains to be defined, as few studies have
correlated in vitro resistance with clinical response.
Development of pneumococcal resistance to penicillin G in
vitro as well as in vivo in a mouse model was reported soon
after the introduction of penicillin in 1943 [4]. However, it was
only in 1967 that decreased susceptibility to penicillin was first
reported with regard to human clinical isolates of pneumococci,
and strains associated with even higher MICs of penicillin as
well as resistance to many other agents have been recognized
since 1977 [1, 3]. Resistant pneumococci have now been reported from all continents and are the predominant pathogens
m some areas.
Since pneumococci are not recovered in most cases of infection, comprehensive antimicrobial susceptibility patterns are
not available. In the United States, the surveillance program
of the Centers for Disease Control and Prevention, surveying
systemic isolates in cases of severe disease at 13 hospitals in
12 states, has demonstrated an increase in penicillin resistance
rates, from 5% in 1979-1987 (with all but one strain being
intermediately penicillin-resistant) to 6.6% in 1991-1992 (with
1.3% of isolates being associated with MICs of ~2 Jlg/rnL)
[5,6]. In addition, 16.4% of all strains were resistant to at least
one drug class.
A recent report from metropolitan Atlanta documented an
overall pneumococcal penicillin-resistance rate of 25% during
1994; 7% of strains were highly resistant, and a high percentage
CID 1996;23 (October)
Resistant Pneumococci in Central and Eastern Europe
were also resistant to other agents [7]. Other studies have shown
that resistant strains are widespread in many countries, and a
multiply resistant clone of a Spanish type-23F strain has been
identified in France, England, South Africa, and 19 states in
the United States. The increased frequency of resistant pneumococci is typified by the experience in Spain, where the incidence
of these strains rose from 8.7% during 1979-1981 to >44%
in 1995 and high-level resistance had risen to nearly 17% by
1990 [8].
The aim of this study was to obtain data on the susceptibility
of pneumococci in Central and Eastern Europe, as little data
outside of Hungary has been reported in the English-language
literature [9]. The approach was a survey of nasopharyngeal
carriers among children under 5 years of age. The study was
standardized by use of a common protocol and by the provision
of common reagents from commercial sources as well as a set
of strains for quality assurance to each center participating in
the study.
Materials and Methods
Children who were hospitalized or attending day-care centers
or outpatient clinics were studied from the period of June 1993
to October 1994. Informed consent was obtained from participants' parents or guardians.
For detection of pneumococcal carriers, nasopharyngeal
swabs were collected with calcium alginate swabs on flexible
aluminum shafts (Fisher Scientific, Pittsburgh). Specimens
were obtained pernasally (with the shaft curved) or via the
mouth (with the last 2 cm of the shaft bent at a 135° angle).
Swabs were plated immediately after collection onto trypticase soy agar with 5% sheep blood and gentamicin (5 p,g/mL)
[2]. Plates were then incubated at 35°C in 5%-10% CO2 for
up to 48 hours, and a-hemolytic colonies were subcultured
onto trypticase soy agar with 5% sheep blood; optochin disks
were placed on the inocula. Strains inhibited by optochin (inhibition zones of> 14 mm) were identified as S. pneumoniae,
and identification was confirmed by bile solubility.
Antimicrobial susceptibility testing was performed with disk
diffusion on Mueller-Hinton agar supplemented with 5% sheep
blood [2, 10]. Growth from a fresh overnight subculture was
suspended in saline and adjusted to a density equal to a 0.5
McFarland standard. Plates were inoculated by the streaking
ofthe entire plate in three directions with a cotton-tipped swab.
Disks containing 1 p,g of oxacillin (to screen for susceptibility
to penicillin G), 15 p,g of erythromycin, 30 p,g of tetracycline,
30 p,g of chloramphenicol, and 25 p,g of trimethoprim-sulfamethoxazole (TMP-SMZ) were then placed on each plate.
Plates were incubated overnight as described above, and zones
of inhibition were interpreted as shown in table 1.
For the purpose ofthis report, both strains that are intermediately resistant to penicillin and that are resistant to penicillin
are declared as resistant. Furthermore (see below), it is possible
that occasional susceptible strains were classified as resistant
713
Table 1. Interpretation of inhibition-zone sizes for antimicrobials
tested against S. pneumoniae isolates.
Agent for which
susceptibility of
isolate was
tested
Disk content
(J.Lg)
Susceptible
Penicillin
Erythromycin
Chloramphenicol
Tetracycline
TMP-SMZ
I (oxacillin)
15
30
30
25
;.20
;.20
;.22
;.20
;.20
Zone size (mm) for isolates of
indicated susceptibility
Intermediate
",,19
NA
NA
NA
16-19
Resistant
",,19
",,19
",,21
",,19
",,15
NOTE. Both strains that were intermediately resistant to penicillin and that
were resistant to penicillin were considered resistant. NA = not applicable.
with use ofthe oxacillin-disk screening method. MICs of penicillin were determined for selected strains by agar dilution
methodology [2]. Serotyping was performed by the capsulequellung method with use of antisera from the Statens Seruminstitut (Copenhagen).
Investigators were provided with a panel of 12 pneumococcal
strains and were required to document proficiency in susceptibility testing of these strains prior to starting the study. Only
centers with antisera performed serotyping.
Results
Twelve quality-control strains were sent to each center for
susceptibility testing. These strains had varying susceptibilities
to the study agents and varied resistance patterns. Four strains
were susceptible to penicillin, 8 to erythromycin, 8 to tetracycline, 9 to chloramphenicol, and 5 to TMP-SMZ. Strains were
tested at each center by disk diffusion, according to the study
protocol. Zone diameter categorical interpretations were correctly determined for all strains in all centers.
Nine hundred and fifty-four children in six countries
(69-253 per country) were enrolled in the study. The sources
of participants included children's hospitals, day-care centers,
and outpatient clinics. Age distribution is shown in table 2.
The overall carriage rate of pneumococci was 27.0%, with
considerable variation between countries (table 3). The carriage
rate was highest in the Romanian center (60.9%) and lowest
in Russia (5%). The rate also was high in Bulgaria (47.7%).
No penicillin-resistant strains were found in centers in the
Czech Republic, Poland, and Russia, while 36.1%-92.9% of
strains from Bulgaria, Romania, and the Slovak Republic were
penicillin-resistant. Most of the penicillin-resistant strains were
resistant to one or more other classes of agent. Multiple-resistance patterns were found, and the most frequent are shown in
table 4. It is interesting that 15 of the 39 penicillin-resistant
strains from Romania had the same pattern of resistance (to
penicillin, erythromycin, tetracycline, and TMP-SMZ) and that
13 were resistant to penicillin, erythromycin, chloramphenicol,
Table 2. Age distribution of the 954 children screened for pneumococcal carriage in the multicenter study.
No. of children per age group (y)
Site of center
Bulgaria (Sofia)
Czech Republic
(Prague)
Poland
(Warsaw)
Romania
(Bucharest)
Russia
(Moscow)
Slovak Republic
(Bratislava)
All centers
em
Appelbaum et aL
714
<I
I to <2
2 to <3
;;;.3
Total
74
77
47
18
216
32
55
13
0
100
67
46
64
39
216
0
43
26
0
69
31
57
12
0
100
88
292
72
350
68
230
25
82
253
954
tetracycline, and TMP-SMZ. In addition, many strains in Poland that were not resistant to penicillin were resistant to tetracycline, and many Slovak and Bulgarian strains were resistant
to TMP-SMZ (table 5).
Four serotypes (6, 14, 19, and 23) accounted for almost all
penicillin-resistant pneumococcal strains: types 6 and 14 were
common in Romania, while type 19 predominated in the Slovak
Republic (table 6). However, serotypes did not correlate well
with resistance patterns: the commonest pattern of resistance
(to penicillin, erythromycin, tetracycline, and TMP-SMZ) in
Romania included strains of all four common serotypes. MICs
of penicillin for selected strains showed considerable variation
in level of resistance, from 0.12 p,g/mL to 8.0 p,g/mL; most
MICs were> 1.0 p,g/mL.
Discussion
Resistance of pneumococci to ,B-lactam drugs has been well
documented to be due to multistep changes in penicillin-binding proteins, resulting in strains associated with a spectrum of
MICs rather than a bimodal distribution of either full suscepti-
Table 3.
bility or high resistance [1, 2]. As a result, for example, MICs
of penicillin can be as low as 0.008 p,g/mL for fully susceptible
strains and as high as 64 p,g/mL for the most resistant strains
described to date. MICs of all ,B-lactam drugs vary in the same
manner, and the range of MICs varies among the different
agents as well.
Therefore, therapy for infections caused by ,B-lactam-resistant strains is influenced by the level of ,B-lactam resistance,
the site of infection, and the route and frequency of administration, as well as the amount of the agent administered. This has
resulted in problems in the design of a universal interpretation
system applicable to all pneumococcal infections. For example,
meningitis-causing strains for which the MICs are 1.0 p,g/mL
are clearly clinically resistant to high-dose intravenous penicillin G, but nonmeningeal strains are clinically susceptible to it
[11]. The clinical response to most oral ,B-lactam drugs in the
treatment of pneumococcal otitis media and other respiratory
infections caused by penicillin-resistant strains (MICs, ;;;;.0.l25
p,g/mL) is generally unknown, as studies have been performed
on patients with infections caused by penicillin-susceptible
strains.
Thus, the clinical significance of penicillin-resistant pneumococci remains problematic, although in a few reports some
clinical correlations were suggested [2, 11, 12]. For example,
MIC breakpoints for cefotaxime and ceftriaxone that are relevant to meningitis have been established, and breakpoints for
oral cefuroxime axetil and oral amoxicillin that are relevant to
otitis media have been established by the National Committee
for Clinical Laboratory Standards (NCCLS). The breakpoints
for these four agents are as follows: ~0.5 p,g/mL, susceptible;
1 p,g/mL, intermediate; and ;;;;.2 p,g/mL, resistant. Breakpoints
for imipenem have also been determined (although little
clinical validation is available): ~0.12 p,g/mL, susceptible;
0.25-0.5 p,g/mL, intermediate; and ;;;;.1 p,g/mL, resistant [13].
It is well known that some strains for which the MIC of
penicillin is 0.06 p,g/mL yield oxacillin zones of <20 mm [2].
However, such strains are rare in Central and Eastern Europe
[9], and any such strains would have been considered resistant
in our study. Interpretation of susceptibility of agents other
Pneumococcal carriage rate among the 954 children in the multicenter study.
No. (%) of carriers
Site of center
Bulgaria (Sofia)
Czech Republic (prague)
Poland (Warsaw)
Romania (Bucharest)
Russia (Moscow)
Slovak Republic (Bratislava)
All centers
Total
103
12
35
42
5
61
258
(47.7)
(12.0)
(16.2)
(60.9)
(5.0)
(24.1)
(27.0)
1996;23 (October)
With penicillinsusceptible isolates
60
12
35
3
5
39
154
(58.3)
(100)
(100)
(7.1)
(100)
(63.9)
(59.7)
With penicillinresistant isolates
43 (41.7)
0
0
39 (92.9)
0
22 (36.1)
104 (40.3)
CID 1996;23 (October)
Resistant Pneumococci in Central and Eastern Europe
715
Table 4. The most common resistance patterns of penicillin-resistant pneumococcal strains in the
multicenter study.
No. of isolates with indicated resistance pattern
Site of center
Bulgaria (Sofia)
Czech Republic (Prague)
Poland (Warsaw)
Romania (Bucharest)
Russia (Moscow)
Slovak Republic (Bratislava)
Total
Pen
43
0
0
39
0
22
4
0
0
0
0
12
Pen, TMPSMZ
Pen, Em, Tet,
TMP-SMZ
Pen, Em,
Tet
Pen, Em, Chi,
Tet, TMP-SMZ
10
0
0
0
0
0
15
0
0
13
0
0
3
0
0
10
0
0
0
4
13
0
0
NOTE. Chi = chloramphenicol; Em = erythromycin; Pen = penicillin G; Tet = tetracycline.
than {3-lactam drugs is much less problematic, as bimodal distributions of susceptible and resistant strains are found with
testing of macrolides, tetracyclines, chloramphenicol, TMPSMZ, and rifampin. However, the outcome of meningitis
caused by chloramphenicol-susceptible strains has been shown
to be associated with susceptibility to penicillin; the clinical
response to chloramphenicol is poorer in infections caused by
penicillin-resistant strains [12].
Zone-diameter interpretations used in this study for erythromycin, tetracycline, chloramphenicol, and TMP-SMZ [10] differ slightly from those recently recommended by the NCCLS
[13]. However, because of the bimodal distribution of strains
against these compounds [2, 10], few if any truly intermediate
strains occur. We therefore did not include an intermediate
category for these agents, and any intermediate strains were
included in the resistant group.
This study highlights the remarkable variation in resistance
patterns in the countries in which studies were conducted, as
many resistance patterns and serotypes were found. Of note is
the lack of resistance to penicillin in the areas studied in the
Czech Republic, Poland, and Russia, while such resistance is
widespread in Bulgaria, Romania, and the Slovak: Republic.
It is also of interest that the countries in which the rates of
Table 5.
pneumococcal carriage among children were highest also have
the highest rates of resistance. The reason for these differences
is unknown but may be associated with differences in antimicrobial availability, route of administration, and dosage.
Antibiotic-resistant pneumococci have been reported from
Eastern and Central Europe since 1977, when four cases of
meningitis were reported from Iasi, Romania, by Vitta et a1.
[14]. The MIC of penicillin was 4 p,g/mL for three strains and
0.4 p,g/mL for the fourth; these strains were of types 3, 4, 12,
and 14, and the type-4 strain was resistant to chloramphenicol
and tetracycline as well.
Further reports from Romania documented that 4 (0.8%) of
498 strains isolated during 1974-1981 from throughout Romania
were penicillin-resistant; the resistant strains were of types 3, 12,
and 19 (two strains), and the MICs of penicillin were 0.6, 3, and
12p,g/mL [15]. Resistance to erythromycin, chloramphenicol, and
tetracycline was also found, and some strains were multiresistant.
Thirty percent of 290 pneumococci isolated in 1991-1992
in Romania were penicillin- and erythromycin-resistant, and
the MICs of penicillin were 0.3-16p,g/mL (for many strains,
3 p,g/mL) [16]. Resistant serotypes from this period were 6, 14,
and 19. Four of sixteen strains recovered from nasopharyngeal
specimens from hospitalized children in 1991 in Sinaia, Roma-
Resistance of penicillin-susceptible pneumococcal strains to other agents.
No. of isolates resistant to indicated agent(s)
Site of center
Bulgaria (Sofia)
Czech Republic (Prague)
Poland (Warsaw)
Romania (Bucharest)
Russia (Moscow)
Slovak Republic (Bratislava)
All centers
Total
Tet
Chi, Tet
60
12
35
3
5
39
154
0
0
21
0
0
0
22
3
0
0
2
6
NOTE. ChI = chloramphenicol; Em = erythromycin; Tet = tetracycline.
TMP-SMZ
7
0
0
0
11
19
Em, Chi,
TMP-SMZ
12
0
0
0
0
0
12
716
Table 6.
Appelbaum et al.
Serotypes of selected penicillin-resistant pneumococcal
strains.
No. of isolates of indicated serotype
Site of center
6
Bulgaria (Sofia)
Czech Republic (Prague)
Poland (Warsaw)
Romania (Bucharest)
Russia (Moscow)
Slovak Republic (Bratislava)
All centers
3
0
0
11
0
3
17
14
19
23
0
0
14
0
3
18
4
0
0
5
0
16
25
5
0
0
5
0
6
16
nia, were penicillin-resistant, and most strains also were resistant to other agents [17].
In Slovakia, nine resistant strains from CSF, pleural fluid,
and middle-ear and respiratory tract specimens were reported
by investigators in Bratislava in 1983 [18]. These strains were
of types 6 and 14, had penicillin MICs of 4-8 jlg/mL, and
were all erythromycin- and tetracycline-resistant. Five were
also chloramphenicol-resistant.
In the Topolcany district of Slovakia during 1985-1991,
252 penicillin-resistant strains were isolated [19]. Of 116
strains tested further, 115 had penicillin MICs of 4-16
jlg/mL and most were of type 14 and resistant to erythromycin,
tetracycline, and chloramphenicol; there was variable susceptibility to TMP-SMZ. Two distinct resistant clones have also
been reported from Slovakia and the Czech Republic from a
collection of 72 strains. One clone, containing 17 of these
strains, was of type 14 and had a penicillin MIC of 8 p,g/mL,
while another clone (of 15 strains) was of type 19 and had
penicillin MICs of 1-4 jlg/mL [20].
In Bulgaria during 1991-1993, 24% of 296 strains were
penicillin-resistant (MICs, 0.25-8 jlg/mL). Serotypes ofpenicillin-resistant strains were 6, 9, 14, 19, and 23, and many
strains were multiresistant, with patterns such as resistance to
penicillin and TMP-SMZ, penicillin/erythromycin/tetracycline,
and many others [21]. Some of the strains reported by Setchanova [21] were included in the current study.
Marton [9] reported on an epidemiologic survey of resistance
to penicillin in pneumococcal strains collected from several
Hungarian laboratories between 1988 and 1989. An overall
prevalence of 58.0% was found, with a significantly higher rate
among children (69.2%) than among adults (44.0%). Penicillinresistant strains were more frequently resistant to tetracycline,
erythromycin, TMP-SMZ, and chloramphenicol than were penicillin-susceptible strains. Serotypes of penicillin-resistant
strains were predominantly 6, 19, and 23.
The value of establishing resistance patterns of pneumococci
with comparable, standardized methods in Eastern and Central
Europe has been well documented in this study, as considerable
differences were found between the centers in these countries.
The centers in Romania, Bulgaria, and Slovakia should now
cm 1996;23 (October)
be regarded as having a high prevalence of resistant pneumococci. The absence of resistant strains in the Czech Republic
(Prague), Poland (Warsaw), and Western Russia (Moscow) in
the current study is also a valuable baseline for monitoring
changes in this situation.
It is important to note that data in this study were obtained
from a limited number of centers, in primarily urban areas.
While the data may be representative of the cities where they
were obtained, extrapolation to the entire countries cannot be
made, and more extensive surveys are required.
Surveys of nasopharyngeal carriage of pneumococci in children are important in determining which strains are prevalent
in each community, as these are the strains responsible for
otitis media, of which a microbiological diagnosis is rarely
made. In addition, susceptibility testing of isolates from sterile
sources should also be performed to guide therapy in individual
cases as well as to provide epidemiologic data. Additional
epidemiologic data, as well as data from clinical trials, are
essential to determine appropriate empirical management for
pneumococcal infections in this era of rapidly increasing pneumococcal resistance to currently available agents.
Acknowledgment
The authors thank Dr. Robert Charnas for his contributions to
the realization of the study.
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