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Journal of Medical Microbiology (2003), 52, 981–987
Characterization of German penicillinnon-susceptible serotype 23F pneumococci usingmultilocus sequence typing
Ralf Rene´ Reinert,1 Stefanie Muckel,1 Adnan Al-Lahham,1 Brian G. Spratt,2Angela B. Brueggemann,3 Regine Hakenbeck4 and Rudolf Lu¨tticken1
1Institute of Medical Microbiology, National Reference Center for Streptococci, University Hospital,
2Department of Infectious Disease Epidemiology, Imperial College School of Medicine, St Mary’s
3Department of Microbiology, University of Oxford, and Department of Public Health & Primary Care,
John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK
4Institute of Microbiology, University of Kaiserslautern, Germany
Three nationwide multicentre studies (n ¼ 5071) showed an increase in antibiotic resistance inpneumococci in Germany. Serotype 23F was the predominant serotype (n ¼ 45, 22·4 %), followedby 6B (n ¼ 30, 14·9 %) and 9V (n ¼ 19, 9·5 %). Multilocus sequence typing was used tocharacterize 45 serotype 23F strains with reduced penicillin susceptibility. The Spanish23F-1 clone[profile 4-4-2-4-4-1-1, sequence type (ST) 81] contributes significantly to the emergence ofpenicillin resistance in Germany (n ¼ 21, 46·7 % of all penicillin non-susceptible serotype 23Fisolates). Isolates of ST 277 (profile 7-13-8-6-6-12-8), which has been found previously in theNetherlands, are also observed, particularly in western Germany (n ¼ 8, 17·8 %). A high proportionof strains (n ¼ 11, 24·4 %) have sequence types that have not been reported to date from other
countries (STs 353–362). The major penicillin-resistant clones are present in Germany, a country
with relatively low levels of â-lactam resistance.
the same period, outpatient pneumococcal isolates werecollected prospectively by 19 clinical microbiology labora-
Penicillin non-susceptible Streptococcus pneumoniae strains
tories in Germany between November 1998 and April 1999
were first reported from Germany in 1992
(study C), with 6·8 % (n ¼ 65) of 961 pneumococcal strains
1992). Within the last 5 years, an increase in both penicillin
showing reduced susceptibility to penicillin
and macrolide resistance has been observed among S.
pneumoniae in Germany, as documented by three surveil-lance studies performed by the National Reference Center for
Multilocus sequence typing (MLST) is a recently developed
Streptococci. In a nationwide study of invasive pneumococ-
technique that produces unambiguous molecular typing
cal disease in adults (study B), the proportion of strains with
data that can be transmitted electronically via the Internet
reduced susceptibility to penicillin (MIC > 0·12 ìg mlÀ1)
increased from 1·8 % in 1992 to 5·8 % in 2000
procedure, nucleotide sequences of $ 450 bp internal frag-
2002). In an ongoing population-based nationwide surveil-
ments of seven housekeeping genes are determined for each
lance study of invasive disease in children (study A), which
isolate. Different sequences at each locus are assigned as
started in 1997 and covered all paediatric hospitals and
distinct alleles and, for each isolate, alleles at the seven loci
clinical microbiology laboratories in Germany, ten strains
define the allelic profile or sequence type (ST). The method is
(3·3 %) were found to be penicillin-intermediate (MIC 0·12–
highly portable, as any laboratory can compare sequences at
1·0 ìg mlÀ1), but no penicillin-resistant strains (MIC
the seven loci in their isolates with those in a central database
. 2 ìg mlÀ1) were detected ., 2000). During
on the World Wide Web (http://www.mlst.net) and obtainthe allelic profile of each isolate. The present study uses thistechnique to analyse clinical strains of penicillin non-
Abbreviations: MLEE, multilocus enzyme electrophoresis; MLST,multilocus sequence typing; NCCLS, National Committee for Clinical
susceptible, serotype 23F S. pneumoniae isolated in Germany
Laboratory Standards; SLV, single locus variant; ST, sequence type.
Strains. Forty-five single patient isolates of serotype 23F pneumococci
that were penicillin-intermediate or -resistant were collected between
1992 and 2000 from three national surveillance projects in Germany
(Table 1). Twelve isolates were recovered from blood, three from
cerebrospinal fluid, 17 from respiratory specimens and 13 from other or
unknown sources. In addition, we scanned the MLST database, which
includes the allelic profiles of over 700 pneumococci, for pneumococcalserotype 23F infections 1998).
Susceptibility testing and serotyping. Susceptibility testing was
performed by using the broth microdilution method as recommended
by the National Committee for Clinical Laboratory Standards
2000). Commercially manufactured plates (Micronaut-S; Merlin Diag-nostics) that contained penicillin, cefotaxime, erythromycin, clin-
damycin, tetracycline and cation-adjusted Mueller–Hinton broth
(Oxoid) plus 5 % lysed horse blood (Oxoid) were utilized. MICs were
determined following incubation at 35 8C for 20–24 h in ambient air. S.
pneumoniae ATCC 49619 was used as a control strain. Isolates werestored at À70 8C on porous beads (Microbank; Mast Diagnostics).
Susceptibility to cotrimoxazole was determined by means of E test strips
(Viva Diagnostica). Current NCCLS interpretive criteria were used to
Pneumococcal strains were serotyped by Neufeld’s Quellung reaction
using type and factor sera provided by the StatensSerum Institut, Copenhagen, Denmark. The serotype distribution of
this collection of penicillin non-susceptible pneumococci was analysed
with reference to all pneumococci collected in three German multi-
DNA purification, PCR protocols and MLST. PCR amplification wascarried out on chromosomal DNA by using an extension time of 30 sand an annealing temperature of 50 8C with Taq polymerase (Roche
Diagnostics). DNA fragments were purified by using the QIAquick
system (Qiagen) and sequencing reactions were carried out in each
direction by using the primers that had been used for initial PCR
amplification. After gel electrophoretic separation of 8 ìl PCR product
in 1 % agarose, amplicons were visualized and their length determinedby using UV transillumination. For gel documentation, the BioDoc-
MLST was carried out as described previously. Briefly, internal frag-
ments of the aroE, gdh, gki, recP, spi, xpt and ddl genes were amplified by
PCR from chromosomal DNA with the primer pairs described by
Assignment of alleles at each locus was carried out with software
available at the pneumococcal MLST website (http://www.mlst.net).
Alleles at each of the seven loci provide the allelic profile of each isolate
and also define their ST. Allelic profiles are shown as the alleles at each of
the seven loci, in the order aroE, gdh, gki, recP, spi, xpt, ddl. Allelic
profiles of the German isolates were compared with each other, as well as
with other isolates in the pneumococcal MLST database, by using
software available at the MLST website.
Detection of resistance genes. For amplification of tetM, primers
originally published by with the sequences 59-
TGGAATTGATTTATCAACGG-39 (positions 2496–2515) and 59-
TTCCAACCATACAATCCTTG-39 (positions 3575–3556) were used.
For amplification of tetO, the primers 59-GGCACAGACCCGTA
TACTGTT-39 (positions 442–462) and 59-TTAAAAGAGGGTCGC
CATCTG-39 (positions 1230–1250) were used al.,
(1997) with the sequences (erm) 59-CGAGTGAAAAAGTACTCAACC-
39 (positions 362–382) and 59-GGCGTGTTTCATTGCTTGATG-39
(positions 978–958) and (mef) 59-AGTATCATTAATCACTAGTGC-39
isolates of this clone. The ST 242 strains showed the MLSB
(positions 57–77) and 59-GTAATAGATGCAATCACAGC-39 (posi-
type of resistance (erm-positive), whereas the SLV (ST 362)
Two strains in the German strain collection (RKI 530 and
RKI 531) belong to ST 142, which is defined by a single
Serotyping data were available for 201 representative peni-
penicillin-resistant serotype 23F strain (M 402) that was
cillin non-susceptible strains of pneumococci collected in
isolated in 1998 in Spain. ST 142 is a SLV of ST 277, which is
three German multicentre studies (Table 1) .,
widespread in Germany. Interestingly, both ST 142 isolates
were isolated in the same city, Dortmund, in 1999. Strains of
predominant serotype (n ¼ 45, 22·4 %), followed by 6B
ST 142 are susceptible to erythromycin and clindamycin;
(n ¼ 30, 14·9 %), 9V (n ¼ 19, 9·5 %), 19A (n ¼ 18, 9 %),
however, in contrast to the Spanish isolate, they are resistant
19F (n ¼ 17, 8·5 %), 14 (n ¼ 14, 7 %), 6A (n ¼ 9, 4·5 %), 15B
(n ¼ 6, 3 %) and others (n ¼ 43, 21·4 %).
One strain (RESP 798; ST 355) is an SLV to the high-level
demographic, antimicrobial susceptibility and
cephalosporin-resistant serotype 23F clone from the US
MLST data for individual strains. The following sequence
(Tennessee23F-4; pneumococcal network, accession no.
types were found: ST 81 (n ¼ 21, 46·2 %), ST 277 (n ¼ 8,
ATCC 51916). In contrast to the Tennessee23F-4 clone, the
17·8 %), ST 242 (n ¼ 2), ST 142 (n ¼ 2), ST 323 (n ¼ 1) and
German isolate remained susceptible to cephalosporins. Five
of the German strains had allelic profiles that were not relatedclosely (SLVs) to any isolates in the MLST database (STs 353,
ST 81 strains were isolated in Germany each year from 1992
to 1999. ST 81 (the ST of the Spanish23F-1 2001) appears to be geographically distributed throughout
Penicillin resistance in pneumococci is now an infectious
Germany and was recovered from both invasive and non-
disease problem of international significance. Penicillin
invasive disease sources. Seven strains among the group of
resistance rates vary even between neighbouring European
German ST 81 strains were high-level penicillin-resistant
extremely high level of penicillin resistance is reported fromsouthern and south-eastern Europe, whereas the level of
Most strains (n ¼ 15, 71·4 %) of ST 81 were susceptible to
pneumococcal â-lactam resistance remains low in Germany
erythromycin (MIC <0·25 ìg mlÀ1), but 90 % of isolates (19
of 21) were resistant to tetracycline (MIC > 4 ìg mlÀ1, all
Netherlands ., 1997), the northern European
tetM-positive). All German isolates of the Spanish23F-1 clone
had the typical allelic profile of this clone (ST 81).
The second most prevalent penicillin-resistant 23F clone in
MLST now offers an excellent tool for unambiguous char-
Germany is ST 277. Eight strains (17·8 %) had ST 277 and
acterization of pneumococci by sequencing approximately
another four were very closely related, differing from the
450 bp fragments of seven housekeeping loci
allelic profile of this ST at only a single locus [single locus
Spratt, 1998). MLST is particularly useful for comparing
variants (SLVs)]. Only one isolate of ST 277 can be found in
penicillin- and multiresistant isolates recovered in one
the MLST database; this is a penicillin-resistant serogroup 23
geographical region with those described elsewhere
strain isolated in the Netherlands (strain JJ279-23). Strains of
ST 277 were mostly isolated (four of eight ST 277 strains) inthe extreme west of Germany, whilst the other strains were
The present study showed serotype 23F to be the most
detected in central and southern Germany. No SLVs of ST 81
predominant penicillin non-susceptible serotype isolated in
were detected, whereas four SLVs of ST 277 (STs 359 and 360,
Germany. Strains from three nationwide surveys were
one each; 361, two strains) were found to be present in
included in the analysis. As study A (population-based study
Germany. All SLVs showed alterations in the xpt gene,
covering all laboratories in Germany) and study B (Table 1)
harbouring allele 6 (strains PS 275 and PS 278; ST 361),
are nationwide studies, the penicillin non-sensitive serotype
allele 20 (RKI 425; ST 359) or allele 65 (RKI 682; ST 360). All
23F pneumococcal strains included in the present analysis
strains of ST 277 and its SLVs were intermediately penicillin-
are representative isolates for invasive pneumococcal disease
resistant and were erythromycin- and tetracycline-suscepti-
in Germany. In contrast, representativeness of the present
analysis for pneumococcal respiratory tract infections maybe limited.
Two strains (PS 1953 and PS 2006) were identified as ST 242;one further strain was an SLV of this ST (RESP 95; ST 362).
Among all serotype 23F pneumococci that were recovered in
Interestingly, ST 242 has only been reported from Taiwan to
Germany from 1992 to 2000, MLST unambiguously demon-
date and is now designated the Taiwan23F-15 clone
strated that the Spain23F-1 clone (ST 81) contributed
1998). The two ST 242 isolates in the German strain
significantly to the spread of penicillin resistance in Ger-
collection were intermediately penicillin-resistant and were
many. Representatives of ST 277 and SLVs of this ST are also
erythromycin- and tetracycline-resistant, as are Taiwanese
observed in Germany. A few isolates were indistinguishable
Table 2. Characteristics of 45 penicillin non-susceptible pneumococcal serotype 23F strains isolated in Germany in 1992–2000, in comparison to international reference strains
Strains from the MLST strain collection are marked with bold face. Abbreviations: F.S., federal state; CLI, clindamycin; CTX, cefotaxime; ERY, erythromycin; PEN, penicillin G; SXT, cotrimoxazole; TET,tetracycline; ST, sequence type; ND, no data; B, Berlin; BA, Bavaria; BW, Baden-Wu¨rttemberg; HE, Hesse; HH, Hamburg; LS, Lower Saxony; NRW, North Rhine–Westphalia; SA, Saxony; TU, Thuringia;BS, bronchial secretion; conj., conjunctiva; CSF, cerebrospinal fluid; Nasop., nasopharynx; PF, pleural fluid; f, female; m, male.
*Identical to strain D306-W-23F in MLST database.
}SLV of ST 37 and ST 33.
ÃÃNew allele seen to date only in Germany.
by MLST (or, in some cases, differed at a single locus) from
antibiotic consumption and development of resistance, it
multiply resistant strains from Taiwan or the USA. Only five
suggested that overuse of certain specific antibiotics is more
strains were not closely related to any in the MLST database.
likely to be related to the increase in resistance. Highprevalence of the Spanish 23F-clone in Germany may also
Data on the epidemiology of pneumococcal disease in
be due to the relatively high consumption of tetracycline,
Germany are scant to date. al. (1995) com-
which was widely used in Germany in the mid-1990s, as this
pared penicillin-resistant S. pneumoniae strains isolated in
23F clone is also tetracycline-resistant. A study
different parts of Germany between 1982 and 1992 with
(2001) has demonstrated that sales of antibiotics varied more
penicillin-resistant isolates, mainly of serogroups 6, 9, 14, 19
than fourfold within Europe; France and Spain had the
and 23, from other European countries. Strains were
highest sales, whereas the Netherlands, Denmark, Sweden
characterized by their serotypes, antibiotic resistance pat-
and Germany had the lowest ). In addition,
terns, penicillin-binding protein properties and multilocus
the relatively high frequency of pre-school children staying at
enzyme electrophoresis (MLEE). Eleven of the 14 penicillin-
home and not at day-care centres may contribute to the lower
resistant German isolates were assigned to five genotypes that
level of antibiotic resistance in Germany.
were also isolated in other countries. The study byal. (1995) included five serotype 23F strains
In conclusion, penicillin non-susceptible serotype 23F iso-
of S. pneumoniae, which are included in the collection of
lates in Germany can be assigned to two major clones, but
strains in this investigation. Serotype 23F strains PS 91-D305
other strains also contribute significantly to resistance. Some
and PS 184-D306, classified as MLEE type 2, were shown by
isolates appear to have not yet been identified outside
MLST to be equivalent to ST 81 (the Spain23F-1 clone).
Germany. The findings of the present study underscore that,
MLEE types 21 and 22 were demonstrated by MLST to be
despite the low level of â-lactam resistance, penicillin-
closely related to and to correspond to ST 277 (strain PS 335-
resistant serotype 23F ST81, which is responsible for high
D310) and an SLV of ST 277 (ST 361, strains PS 275-D307
resistance rates reported from other European countries, is
and PS 278-D308), respectively. These observations under-
present in Germany. Consequently, to preserve the currently
score the high discriminative power of both MLEE and
low antimicrobial resistance levels among clinical isolates of
MLST and document that both methods lead to identical
S. pneumoniae, prudent usage of antimicrobials in Germany
results when investigating clonal relatedness of pneumococ-
of the major Spanish clones of penicillin-resistant andmultiply antibiotic-resistant S. pneumoniae strains, includ-
Presented in part at the 11th European Congress of Clinical Micro-
ing previously characterized isolates of the Spanish23F-1
biology and Infectious Diseases, Istanbul, Turkey, 1–4 April 2001. The
clone Fifteen out of 16 of these isolates
authors thank Nelli Neuberger, Maria Lemperle and Claudia Cremer for
had an identical allelic profile (ST 81, profile 4-4-2-4-4-1-1),
excellent technical assistance and Susan Griesbach, Mu¨nster, for copyediting. The study was supported by grant RKI-415/1369235 from the
which was considered to be the typical allelic profile of the
German Minister of Health (Bundesminister fu¨r Gesundheit) and by a
CAP-net grant [German Ministry of Education and Research (Bundes-ministerium fu¨r Bildung und Forschung)].
Data from this study clearly document that most penicillinnon-susceptible strains are closely related to those found inSpain. Results of our study also underscore that travelling
may contribute to the spread of resistance. Germans spent82·6 billion DM (38·4 billion US$) on travel in 1998. For
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Lab Activity for Chapter 9 This laboratory period will be spent working on a few problems in context exercises from Chapter 9. Work on each problem as if you were doing it for homework. Use the computer to obtain graphs andnumerical data values, and to find solutions to equations using graphical and numerical methods. Then, either copy the computer results onto your homework paper by hand, or m