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Antimicrobial Agents and Chemotherapy, May 2001, p. 1578-1580, Vol. 45, No. 5
Department of Microbiology, The Chinese
University of Hong Kong, Prince of Wales Hospital,
Shatin,1 and Pamela Youde Nethersole
Eastern Hospital,2 Hong Kong, China
Received 17 August 2000/Returned for modification 31 January
2001/Accepted 15 February 2001
Erythromycin resistance rates among penicillin-susceptible
Streptococcus pneumoniae were 38 and 92% among
penicillin-intermediate and -resistant S. pneumoniae
isolates from Hong Kong, respectively, and 27% (43 of 158) of the
isolates showed the MLSB phenotype, and the majority
carried the ermB gene; 73% (115 of 158) displayed the M
phenotype, and all possessed the mef gene. The
MLSB phenotype was predominant in penicillin-susceptible,
macrolide-resistant isolates and in penicillin-nonsusceptible isolates
of serotype 6B, whilst the M phenotype was predominant in
penicillin-intermediate or -resistant isolates belonging to serotype
23F or 19F. Extensive spread of clones of drug-resistant pneumococci
has led to the widespread presence of macrolide resistance in S. pneumoniae in Hong Kong.
Macrolide resistance among
Streptococcus pneumoniae has increased in many countries,
such as the United States (2, 3) and United Kingdom
(4). Two principal mechanisms of macrolide resistance have
been described (10); target modification is mediated by an
rRNA erythromycin resistance methylase and coded by the ermB
(ermAM) gene. These organisms express the MLSB
phenotype and show broad cross-resistance to macrolides, lincosamides,
and streptogramin B antibiotics. The M phenotype involves an active efflux pump, which removes only 14-membered and 15-membered macrolides from the bacterial cell (14). The determinant was
identified to be the mef (mefE) gene
(15). Isolates showing the M phenotype are susceptible to
clindamycin and streptogramin B. In 1999, two further mechanisms were
described, one mediated by alterations in the L4 ribosomal protein, and
another due to mutation in the 23S rRNA (15a). The incidence of
erythromycin resistance in pneumococci at the Prince of Wales Hospital,
Hong Kong, has risen from 22% in 1993 to 42% in 1997 (6). The likely explanation is the dissemination of a few
clones of multidrug-resistant S. pneumoniae in the Hong Kong
population as previously described (5). The objective of
the present study was to investigate the prevalence and distribution of
the macrolide resistance determinants in isolates of S. pneumoniae in Hong Kong. In addition, the relationships between
these determinants, penicillin susceptibility, and their serotypes were
also determined.
A total of 197 strains of S. pneumoniae isolated at the
Prince of Wales Hospital and Pamela Youde Nethersole Eastern Hospital, Hong Kong, from 1994 to 1998 were studied. Approximately 25% were invasive isolates from blood and cerebrospinal fluid, and the remainder
were from sputum which had reduced penicillin susceptibility by the
1-µg oxacillin disk method. The two hospitals are located in the
northern and southern parts of Hong Kong, and the isolates were thus
deemed to be representative of strains from patients with pneumococcal
infections requiring hospital admissions in Hong Kong. The
penicillin-intermediate or -resistant isolates had previously been well
characterized by molecular typing using pbp gene profiles
and pulsed-field gel electrophoresis (PFGE) (5).
MICs of penicillin, erythromycin, clarithromycin, and clindamycin were
determined by the agar dilution method (8). Inocula of
104 CFU/spot were incubated at 35°C for 18 h on
Mueller-Hinton agar supplemented with 5% defibrinated horse blood.
S. pneumoniae ATCC 6315 and 49619 were included as controls.
Inducible resistance to clindamycin was detected by the method
previously described (11). Conserved primer sets were
chosen to amplify a 640-bp fragment of the erm methylase
genes as follows: ermA, 5'-TCTAAAAAGCATGTAAAAGAA-3' and 5'-CTTCGATAGTTTATTAATATTAGT-3';
ermB, 5'-GAAAAGGGTACTCAACCAAATA-3' and 5'-AGTAACGGTACTTAAATTGTTTAC-3'; and
ermC, 5'-TCAAAACATAATATAGATAAA-3' and
5'-GCTAATATTGTTTAAATCGTCAAT-3'. A 1.2-kb fragment of the
mef gene in S. pneumoniae was amplified using
primer pair 5'-GAAAAATACAACAATTGGAAAC-3' and
5'-AATCTAATTTTCTAACCTCA-3'. PCRs were performed on the
OmniGene DNA thermal cycler (Hybaid) using an initial denaturation at
94°C for 5 min followed by 35 cycles of amplification at 94°C for
30 s, 50°C for 30 s, and 72°C for 90 s. A final
elongation step was performed at 72°C for a further 7 min. All the
strains were serotyped by the chessboard agglutination or the quellung
reaction method using Pneumotest antisera (Statens Seruminstitut,
Copenhagen, Denmark).
A total of 80% (158 of 197) of the S. pneumoniae strains
were resistant to erythromycin with a MIC of
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.5.1578-1580.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Macrolide Resistance in Streptococcus
pneumoniae in Hong Kong
ABSTRACT
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1.0 µg/ml, 27% (43 of
158) showed an MLSB phenotype with erythromycin and
clindamycin 50% and 90% MICs (MIC50 and
MIC90) of >64 µg/ml; 73% (115 of 158) showed resistance
to erythromycin alone, with MIC50 and MIC90 of
8.0 µg/ml. The MICs of the antibiotics tested for these two groups
are shown in Table 1. Of the isolates
expressing the MLSB phenotype, only the ermB
gene was detected in 93% of the isolates by PCR. Three isolates were
repeatedly negative on testing for ermA, -B, or
-C, and hence perhaps other mechanisms of macrolide resistance exist. All the isolates expressing the M phenotype were
positive for the mef gene by PCR. Table
2 shows the serotypes of S. pneumoniae in relation to penicillin and macrolide susceptibility. Among the 42 penicillin-susceptible isolates, 16 (38%) isolates were
resistant to erythromycin with a MIC of 1.0 µg/ml, and 62.5% of
the penicillin-susceptible, macrolide-resistant isolates showed the
MLSB phenotype. However, of the 155 penicillin-intermediate or -resistant isolates examined, 142 (92%) isolates were resistant to
erythromycin, 107 of 142 (75%) of these belonged to the M phenotype, whilst the remainder (35 of 142, 25%) belonged to the MLSB
phenotype, with a MLSB/M phenotype ratio of 1:3. The
MLSB phenotype was mainly associated with serotype 6B
(P > 0.001, chi square test), whilst the M phenotype
was predominant in isolates expressing serotype 23F or 19F
(P > 0.001, chi square test).
TABLE 1.
MICs of 197 Hong Kong S. pneumoniae isolates
for four antibiotics by macrolide and lincosamide susceptibility
phenotypes
TABLE 2.
Serotypes of S. pneumoniae in relation to
penicillin and macrolide susceptibilitya (197 isolates)
The study indicated a high percentage of erythromycin resistance among clinical isolates of S. pneumoniae in Hong Kong, with 38% in penicillin-susceptible pneumococci and 92% of penicillin-intermediate or -resistant isolates. Our previous study (5) showed that these strains belonging to 19F and 23F had identical pbp profiles and PFGE fingerprints and belong to clones indistinguishable from that belonging to the Spanish 23F clone. Similarly, isolates belonging to serotype 6B had unique pbp profiles and identical PFGE fingerprints to that of the Spanish 6B clone (5). The data further support the hypothesis that the dissemination of particular clones of S. pneumoniae of reduced penicillin susceptibility has been responsible for the spread of macrolide resistance determinants in Hong Kong. In Canada and the United States, the M phenotype was prevalent in 55.8 and 71%, respectively, of the macrolide-resistant pneumococci (3, 7). In contrast, the MLSB resistance mechanism predominates in European countries, and the mef gene was only found in 1, 5.8, and 9% among resistant pneumococci in France (P. Angot, M. Vergnaud, and R. Leclerq, Program Abstr. 39th Intersci. Conf. Antimicrob. Agents. Chemother. abstr. 1221, 1999), Italy (9), and Belgium (1) respectively. Macrolide resistance has been reported to be high among pneumococci in Asian countries (13), but the distribution of these macrolide resistance determinants is not known. As in Hong Kong, penicillin- and multidrug-resistant S. pneumoniae clones belonging to serotypes 19 and 23 disseminated widely in the last decade in Taiwan (12), Korea (17), and Japan (18), and it is likely that the mef genes are prevalent among the penicillin-resistant pneumococci in these countries too.
The 14- and 15-membered macrolides have poor activity against Hong Kong clinical isolates of S. pneumoniae, particularly against penicillin-intermediate or -resistant strains. This study supported the widespread dissemination of the macrolide resistance determinants, particularly the mef gene, which occurred with the spread of penicillin-nonsusceptible pneumococcal clones in Hong Kong.
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ACKNOWLEDGMENTS |
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The work described in this paper was fully supported by grants from the Research Grants Council of the Hong Kong Special Administrative Region (Project Nos. CUHK 4215/97M and 4331/99M).
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FOOTNOTES |
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* Corresponding author. Mailing address: Dept. of Microbiology, Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong, China. Phone: (852) 2632 2306. Fax: (852) 2647 3227. E-mail: margaretip{at}cuhk.edu.hk.
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Antimicrobial Agents and Chemotherapy, May 2001, p. 1578-1580, Vol. 45, No. 5
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.5.1578-1580.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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