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JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 2006, p. 4625–4627 0095-1137/06/$08.00ϩ0 doi:10.1128/JCM.01740-06Copyright 2006, American Society for Microbiology. All Rights Reserved.
Emergence of a Unique Multiply-Antibiotic-Resistant Streptococcus pneumoniae
Serotype 7B Clone in Dhaka, Bangladeshᰔ
Streptococcus pneumoniae is a frequent cause of potentially life-threatening infections, such as pneumonia, meningitis, and TABLE 1. MIC ranges, MIC s, and MIC s of 136 S. pneumoniae bacteremia (14). However, the global emergence of antimicro- bial resistance in S. pneumoniae is a serious concern (4). Re- cent data from 12 Asian countries showed high resistance rates (17, 18). We studied prospective resistance to a large number of antimicrobial agents in pneumococcal isolates. We also ana- lyzed macrolide resistance, with an emphasis on resistance genes, molecular epidemiology, and serotype patterns.
From 1999 to 2002, S. pneumoniae isolates (n ϭ 136) from blood and cerebrospinal fluid (CSF) (n ϭ 60) and nasopharynx (n ϭ 76) of children (Ͻ5 years) with pneumonia and meningitis from three hospitals in Dhaka, Bangladesh, were studied.
MICs were determined by CLSI broth microdilution method (1) and by Etest (AB Biodisk, Solna, Sweden). Macrolide re- sistance phenotypes were determined by triple-disk test (11, 12) and macrolide resistance genotypes by a light cycler pro- tocol (15). Isolates were serotyped by antisera (Statens Seru- minstitut, Copenhagen, Denmark). Multilocus sequence typing (MLST) was carried out (2), and two predominant sequence SXT, sulfamethoxazole-trimethoprim. *, MICs were determined by Etest (see text). For ciprofloxacin, a breakpoint of Ͼ4 ␮g/ml was used.
types (ST) were analyzed by use of the eBURST2 program (3).
MIC results for S. pneumoniae (Table 1) showed high rates netically related to this clonal complex (strain 28, ST 113).
of resistance to sulfamethoxazole-trimethoprim (77.9%) and eBURST analysis (Fig. 1) showed ST 1553 and ST 1586 to tetracycline (46.3%). The resistance rates of other drugs were form a pair of SLVs. No other SLVs were found in the MLST low. The rates of multiply-resistant isolates were 27.9% and database (www.mlst.net). An analysis for double-locus variants 11.7% against 2 and Ն3 classes of antibiotics, respectively. Six determined these two ST to be members of a group of 26 ST (4.4%) isolates (Table 2) were resistant to erythromycin A; five represented by 37 isolates with ST 230 as the predicted group of them exhibited the partially inducible iMcLS founder. ST 230 is represented in the MLST database by two (susceptible or intermediate to rokitamycin but developing no erythromycin-sensitive serotype 14 isolates from Denmark and induction resistance to rokitamycin in the presence of eryth- Italy and an erythromycin-resistant serotype 24F isolate, also romycin) and one strain the M phenotype (resistant to eryth- from Italy. Six ST in the group are double-locus variants of romycin, azithromycin, and clarithromycin but susceptible to clindamycin and streptogramin B). Isolates with the iMcLS Our study highlights a high level of tetracycline and sulfa- phenotype were positive for the erm(B) gene, and the isolate methoxazole-trimethoprim resistance in Bangladesh. There is in- displaying the M phenotype was positive for mef(A). Macrol- creasing concern over resistance in pneumococci to sulfamethox- ide-resistant isolates were serotypes 7B (n ϭ 4), 9V (n ϭ 1), azole-trimethoprim, which is recommended by the WHO as a and 18C (n ϭ 1). MLST results of serotype 7B macrolide- first-line drug for treating nonsevere pneumonia. Our study sup- resistant strains established two sequence types: ST 1553 ports the view that this recommendation may not be optimal for (strains 14, 39, and 94) and ST 1586 (strain 61). ST 1586 is a Bangladesh; however, changing to alternative agents, such as single-locus variant (SLV) of ST 1553, and both appear to amoxicillin, is costly (http://www.who.int/child-adolescent-health belong to a single clonal complex. In addition, one isolate was /publications/referral_care/chap3/chap31.htm). Moreover, the a serotype 9V variant of ST 1553 (strain 68), indicating sero- widespread use of sulfamethoxazole-trimethoprim may further type switching. The mef(A)-positive strain was not closely ge- drive the spread of multiply-resistant pneumococcal clones and TABLE 2. Characteristics of macrolide-resistant S. pneumoniae strains isolated from children in Bangladesha a Abbreviations: NP, nasopharynx; ERY, erythromycin A; CLI, clindamycin; PEN, penicillin G; SXT, sulfamethoxazole-trimethoprim; TET, tetracycline; CHL, b ST 1553 showed alleles 6, 5, 2, 17, 6, 22, and 14; ST 1586 showed alleles 43, 5, 2, 17, 6, 22, and 14; and ST 133 showed alleles 7, 2, 1, 1, 10, 1, and 21.
FIG. 1. eBURST analysis of ST of two multiply-resistant serotype 7B strains in this study (ST 1586 and ST 1553) and 2,681 different ST of S. pneumoniae available in the MLST database (www.mlst.net). Single-locus variants are connected by black lines. Double-locus variants are notconnected by lines, with the exception of the double-locus variants of ST 1553 and ST 1586, which are connected by gray lines.
may also select resistance to penicillin G, chloramphenicol, eBURST analysis. The strain exhibiting a mef genotype be- and macrolides. Our observation of a high level of resistance to longs to ST 113. Strains of this clone (global clone 36 of the sulfamethoxazole-trimethoprim in Bangladesh is consistent Pneumococcal Molecular Epidemiology Network [http://www with findings from the mid-1990s (16). In contrast, the rates of .mlst.net; http://www.sph.emory.edu/PMEN]) (10) are serotype resistance to penicillin G, macrolides, and other drugs are 18C and were isolated from meningitis in The Netherlands, the relatively low compared to those described in recent reports United Kingdom, and Spain in the 1980s and 1990s. Interest- from other Asian countries, except India (8, 17, 18). Multiply- ingly, the present report is the first to document macrolide resistant S. pneumoniae is a problem in Bangladesh. Although resistance in an isolate of this particular clone.
an increasing trend of fluoroquinolone resistance has been In summary, our report shows that resistance to beta-lac- reported in Hong Kong, Spain, and Canada (7, 9), a low rate tams, macrolides, and fluoroquinolones in pneumococci is not (2.9%) of ciprofloxacin resistance was observed in our study.
as yet a serious problem in Bangladesh, unlike in many other However, gatifloxacin and moxifloxacin remained active in Asian countries. However, the emergence of a unique multi- vitro, indicating their potential utility in Bangladesh.
ply-resistant serotype 7B clone reiterates the need for contin- The predominance of the MLS phenotype (resistance to all ual surveillance of antimicrobial resistance in pneumococci.
macrolides, lincosamides, and streptogramin B)/erm(B) geno-type in our study is consistent with recent findings from Sri This research protocol was funded by USAID (Washington, D.C.) Lanka, Korea, China, Taiwan, Japan, Spain, Italy, France, and grant number 00097 and by a visiting scientist grant from the German South Africa (6, 9, 15, 17). All strains with the MLS pheno- National Reference Center for Streptococci. ICDDR,B acknowledges type exhibited a partially macrolide-inducible iMcLS pheno- with gratitude the commitment of USAID and the German National Reference Center for Streptococci to the Center’s research efforts.
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Published ahead of print on 27 September 2006.

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