Emergence of a unique multiply-antibiotic-resistant Streptococcus pneumoniae serotype 7B clone in Dhaka, Bangladesh.

Streptococcus pneumoniae is a frequent cause of potentially life-threatening infections, such as pneumonia, meningitis, and bacteremia (14). However, the global emergence of antimicrobial resistance in S. pneumoniae is a serious concern (4). Recent 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 analyzed macrolide resistance, with an emphasis on resistance genes, molecular epidemiology, and serotype patterns. From 1999 to 2002, S. pneumoniae isolates (n 136) from blood and cerebrospinal fluid (CSF) (n 60) and nasopharynx (n 76) of children ( 5 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 resistance phenotypes were determined by triple-disk test (11, 12) and macrolide resistance genotypes by a light cycler protocol (15). Isolates were serotyped by antisera (Statens Seruminstitut, Copenhagen, Denmark). Multilocus sequence typing (MLST) was carried out (2), and two predominant sequence types (ST) were analyzed by use of the eBURST2 program (3). MIC results for S. pneumoniae (Table 1) showed high rates of resistance to sulfamethoxazole-trimethoprim (77.9%) and tetracycline (46.3%). The resistance rates of other drugs were low. The rates of multiply-resistant isolates were 27.9% and 11.7% against 2 and 3 classes of antibiotics, respectively. Six (4.4%) isolates (Table 2) were resistant to erythromycin A; five of them exhibited the partially inducible iMcLSB phenotype (susceptible or intermediate to rokitamycin but developing no induction resistance to rokitamycin in the presence of erythromycin) and one strain the M phenotype (resistant to erythromycin, azithromycin, and clarithromycin but susceptible to clindamycin and streptogramin B). Isolates with the iMcLSB phenotype were positive for the erm(B) gene, and the isolate displaying the M phenotype was positive for mef(A). Macrolide-resistant isolates were serotypes 7B (n 4), 9V (n 1), and 18C (n 1). MLST results of serotype 7B macrolideresistant strains established two sequence types: ST 1553 (strains 14, 39, and 94) and ST 1586 (strain 61). ST 1586 is a single-locus variant (SLV) of ST 1553, and both appear to belong to a single clonal complex. In addition, one isolate was a serotype 9V variant of ST 1553 (strain 68), indicating serotype switching. The mef(A)-positive strain was not closely genetically related to this clonal complex (strain 28, ST 113). eBURST analysis (Fig. 1) showed ST 1553 and ST 1586 to form a pair of SLVs. No other SLVs were found in the MLST database (www.mlst.net). An analysis for double-locus variants determined these two ST to be members of a group of 26 ST represented by 37 isolates with ST 230 as the predicted group founder. ST 230 is represented in the MLST database by two erythromycin-sensitive serotype 14 isolates from Denmark and Italy and an erythromycin-resistant serotype 24F isolate, also from Italy. Six ST in the group are double-locus variants of either ST 1586 or ST 1553 or both. Our study highlights a high level of tetracycline and sulfamethoxazole-trimethoprim resistance in Bangladesh. There is increasing concern over resistance in pneumococci to sulfamethoxazole-trimethoprim, which is recommended by the WHO as a first-line drug for treating nonsevere pneumonia. Our study supports the view that this recommendation may not be optimal for Bangladesh; however, changing to alternative agents, such as amoxicillin, is costly (http://www.who.int/child-adolescent-health /publications/referral_care/chap3/chap31.htm). Moreover, the widespread use of sulfamethoxazole-trimethoprim may further drive the spread of multiply-resistant pneumococcal clones and TABLE 1. MIC ranges, MIC50s, and MIC90s of 136 S. pneumoniae strains isolated in Bangladesh