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Antimicrobial Agents and Chemotherapy, September 2001, p. 2648-2650, Vol. 45, No. 9
MRC/SAIMR/WITS Pneumococcal Diseases Research Unit,
Department of Clinical Microbiology and Infectious Diseases, South
African Institute for Medical Research,
Johannesburg,1 and Microbiology
Department, Drs. du Buisson, Bruinette, and Kramer,
Pretoria,2 South Africa, and Departments
of International Health and Infectious Diseases, Emory University,
Atlanta, Georgia3
Received 23 January 2001/Returned for modification 10 April
2001/Accepted 10 June 2001
We report two South African serotype 6B pneumococcal isolates with
cephalosporin resistance, yet with susceptibility to penicillin. DNA
fingerprinting revealed that they were clonal in origin. pbp 2X and 1A genes showed major alterations
typical of cephalosporin-resistant pneumococci. The pbp
2B gene was completely unaltered, explaining the penicillin
susceptibility of the isolates.
Properties of the pneumococcal isolates studied are shown in Table
1. Antibiotic MICs were determined using
the agar dilution method as specified by the National Committee for
Clinical Laboratory Standards (10). The chromosomal DNAs
of bacteria were fingerprinted using the previously described methods
of pulsed-field gel electrophoresis (7) and BOX-PCR
fingerprinting (5). Chromosomal DNAs were extracted from
bacterial cells, and pbp 2X, pbp 2B, and
pbp 1A genes were amplified from the chromosomal DNAs by PCR
using methods that have been described previously (14).
For pbp gene PCR, primers have been described previously
(12). PCR gene products were fingerprinted using
previously described methods of restriction endonuclease analysis
(14) and were sequenced using the Big Dye Terminator Cycle
Sequencing kit (Applied Biosystems, Foster City, Calif.) and the
Applied Biosystems Model 310 automated DNA sequencer. Altered pbp
2B genes from isolates 8303 and 3191 were independently cloned
into plasmid pGEM-3Zf and were used as a source of transforming DNA.
Isolate 45904 was made competent and was used as recipient in
transformation experiments using the following methods. Bacteria were
cultured in C medium (15) until the mid-exponential phase
(optical density at 620 nm, 0.15) and after the addition of glycerol to
10% were frozen at
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.9.2648-2650.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Emergence of a Pneumococcal Clone with
Cephalosporin Resistance and Penicillin Susceptibility
ABSTRACT
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-Lactam antibiotics inhibit the
growth of pneumococci by inactivating penicillin-binding proteins
(PBPs), which leads to a loss of cell wall synthesis, cell death, and
cell lysis (16). Resistance to -lactams is due to a
complex production of altered PBPs with resultant, decreased antibiotic
affinities (6, 17). Streptococcal "resistance" genes
coding for altered PBPs are acquired from the environment by natural
processes of transformation and are then recombined into the genome of
the pneumococcus (2, 11). Penicillin resistance can be
established by alteration in PBPs 2X, 2B, and 1A (1),
while only altered PBPs 2X and 1A are required for
cefotaxime/ceftriaxone resistance (9). Filipe and Tomasz
(3) showed that a functional MurMN operon is
critical for the expression of penicillin resistance in the pneumococcus. The MurMN operon codes for the MurM and MurN
proteins, which control the biosynthesis of branched-stem structured
cell wall muropeptides. It has recently been shown that in conjunction with altered PBPs, alteration in MurM assists in the development of
-lactam resistance (13). Our present paper reports two
South African serotype 6B pneumococcal isolates with cephalosporin
resistance, yet with susceptibility to penicillin.
70°C in 500-µl aliquots. For transformation,
1 µg of DNA was added to 500 µl of competent cells, which was then
incubated at 30°C for 45 min and at 37°C for 90 min.
Eighty-microliter amounts were then plated onto Mueller-Hinton blood
agar containing penicillin at concentrations ranging from 0.03 to 4 µg/ml, and the plate contents were incubated at 37°C for 48 h.
Transformants grew on agar with penicillin concentrations up to 1 µg/ml. Transformants were picked from 1-µg/ml plates and were
further analyzed.
TABLE 1.
Properties of pneumococcal isolates
Two unique serotype 6B pneumococci were recently isolated in South
Africa, which revealed cephalosporin resistance (cefotaxime and
ceftriaxone MICs, 4 µg/ml; cefuroxime MIC, 8 µg/ml) yet were susceptible to penicillin (MIC, 0.06 µg/ml). Oxacillin disk (1 µg)
screening showed 16-mm zones of inhibition for both isolates. Isolate
45904 was cultured from an ear swab on 30 June 2000 from a 10-month-old
child who presented with an ear infection and perforated eardrum. The
infection was treated with cefaclor. One month prior to this infection,
the child had also suffered from an ear infection which was treated
with cefuroxime. Isolate 53349 was cultured from an ear swab on 21 August 2000 from an unrelated 1-year-old child. This child had suffered
with recurrent middle ear infections and was treated sporadically with
cefpodoxime or amoxicillin/clavulanate over a period of several months
before the specimen was taken. During the month of March 2000, the
child also developed pneumonia, which was treated with ceftriaxone.
Pulsed-field gel electrophoresis and BOX-PCR fingerprinting revealed
identical chromosomal DNA fingerprint patterns for isolates 45904 and
53349, indicating that they were clonal in origin. This was supported
by identical fingerprint patterns for their pbp 2X,
2B, and 1A genes, indicating identical -lactam
resistance genotypes for both isolates. Our investigations revealed no
epidemiological link between the isolates; however, this link cannot be
totally excluded. The pbp 2X, 2B, and
1A genes were sequenced from isolate 45904 and were compared to the sequences of the genes from susceptible laboratory strain R6.
The penicillin-binding-domain-encoding region of PBP 2X revealed 18%
nucleotide sequence divergence, resulting in 39 amino acid mutations in
the protein (Fig. 1). The corresponding
area of PBP 1A revealed 21% nucleotide sequence divergence, resulting
in 41 amino acid mutations in the protein (Fig.
2). However, the pbp 2B gene
was completely unaltered, which would explain the
penicillin-susceptible phenotype of the isolate, since penicillin
resistance is dependent on alterations in PBP 2B. Conservation of
penicillin susceptibility via an unaltered PBP 2B was confirmed by
transformation studies with altered pbp 2B DNA from
penicillin-resistant (MICs, 
4 µg/ml) isolates 8303 and 3191, which
was shown to successfully transform the penicillin MIC for isolate
45904 from 0.06 µg/ml to 2 µg/ml.
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Pneumococci have a total of six PBPs, yet alterations in only two PBPs (PBPs 2X and 1A) are required for cephalosporin resistance (9). Furthermore, the close linkage of pbp 2X and 1A genes on the pneumococcal chromosome is conducive to the transfer of both genes in a single genetic event (4). In the laboratory, one-step transformations have been shown to transform cephalosporin-susceptible strains to MICs of up to 32 µg/ml (9). To the best of our knowledge, isolates 45904 and 53349 are the first pneumococci of this phenotype to be reported that have been isolated in their natural environment and which fulfill laboratory predictions of high cephalosporin resistance emergence coupled with only altered PBPs 2X and 1A. The selective pressure created by cephalosporin therapy has most likely selected the altered pbp genotype in the isolate.
Our study has demonstrated that the pneumococcus can follow various
genetic pathways in its pursuit of resistance to -lactam antibiotics. Alteration in only PBPs 2X and 1A has resulted in the
emergence of cephalosporin-resistant pneumococci in their natural setting.
Nucleotide sequence accession numbers. Sequence data from isolate 45904 appear in the EMBL, GenBank, and DDBJ nucleotide sequence data libraries under the following accession numbers: AF387162 (pbp 2X gene) and AF387163 (pbp 1A gene).
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FOOTNOTES |
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* Corresponding author. Mailing address: MRC/SAIMR/WITS Pneumococcal Diseases Research Unit, Department of Clinical Microbiology and Infectious Diseases, South African Institute for Medical Research, P.O. Box 1038, Johannesburg, 2000, South Africa. Phone: 27-011-4899335. Fax: 27-011-4899332. E-mail: anthonys{at}mail.saimr.wits.ac.za.
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Antimicrobial Agents and Chemotherapy, September 2001, p. 2648-2650, Vol. 45, No. 9
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.9.2648-2650.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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