Dissemination of multiple antibiotic resistant clones of Streptococcus pneumoniae across regions and countries is well documented.1–5 Global clones and their variants, which have spread in Taiwan, include Spain6B-2, England14-9, Taiwan19F-14, Colombia23F-26, Spain23F-1, and Taiwan23F-15.5,6 Recently, Hsieh et al, reported the emergence of invasive serotype 19A isolates in Taiwan among the 2007 invasive pneumococcal isolates.3 To prevent pneumococcal infections, 7-valent pneumococcal conjugate vaccine (PCV7) is being widely used.1,7 PCV7 has significantly reduced invasive pneumococcal diseases (IPD) caused by vaccine serotypes, but serotype 19A has dramatically increased in some countries, but not in others.1,8 In Taiwan PCV7 was not available until October 2005.3 The vaccine is now being used in the private sector, with a low penetration in the pediatric population.3 The aim of this study was to determine the sequence types (STs) of common serotypes that caused IPD in Taiwan after the introduction of PCV7. We also analyzed antimicrobial susceptibility patterns of these isolates.
The 95 IPD isolates were selected for sequence typing and antimicrobial susceptibility testing because their serotype distribution covered the most common 7 serotypes currently circulating in Taiwan.3 All these pneumococcal isolates were identified as described previously by Hsieh et al.6 An IPD isolate was from a positive blood culture or pleural fluid culture from a child with a consolidation pattern upon chest x-ray. IPD also included primary bacteremia without focus defined as a positive blood culture from a child with fever but without a focal lesion. These isolates were collected from Chang Gung Children's Hospital (CGCH) during 2005–2007. The age range of children was from 1 to 9 years, with a median of 4.5 years. Prior to all experiments, the S. pneumoniae isolates were cultivated in trypticase soy agar with 5% sheep blood (in CO2 incubator). Antimicrobial susceptibility to penicillin, ceftriaxone, erythromycin and imipenem was assayed by E-test (AB Biodisk, Solna, Sweden) and interpretation was based on Clinical and Laboratory Standards Institute standards.9 The susceptible, intermediately resistant, and resistant MIC interpretative breakpoints for penicillin (nonmeningitis criteria) were ≤2 μg/mL, 4 μg/mL, and ≥8 μg/mL, respectively.9 Serotyping was performed by latex agglutination and confirmed by Quellung reaction (Statens Serum Institute, Copenhagen, Denmark). All the serotypes were double checked by a PCR method described earlier.10 The nucleotide sequences of 450-bp internal regions from the aroE, gdh, gki, recP, spi, xpt, and ddl genes were amplified by PCR, using the primers, which were described previously.11 The sequences were then compared with those of all the recognized alleles of each gene listed in the pneumococcal multilocus sequence typing (MLST) website database (http://spneumoniae.mlst.net) by BioEdit Sequence Alignment Editor. The Web database (www.mlst.net) was used for assigning allele numbers for particular loci, the STs of isolates were designated on the basis of the resulting allelic profiles.
Among the 95 isolates, 19 sequence types were found, and 27 (28%) isolates from 3 serotypes could be assigned to 4 global clones: Spain6B-2 (ST90, 3 isolates), Taiwan19F-14 (ST236, 14), Spain23F-1 (ST81, 5), and Taiwan23F-15 (ST242, 5) (available at: http://spneumoniae.mlst.net). Majority of serotype 3 isolates belonged to ST180 (Table, Supplemental Digital Content 1, http://links.lww.com/INF/A344). In serotype 6A, ST902 was predominant, while in serotype 6B 6 isolates were assigned to ST76 and 3 to ST90. Among serotype 14, ST876 was found in 7 isolates, followed by ST46 (5 isolates) and ST13 (5 isolates). Majority of 19A belonged to ST320. First clinical isolate of serotype 19A-ST320 was isolated in February 2007 in Taiwan from an 8-year-old patient with bacteremia. In serotype 19F, ST236 was found in 7 isolates and ST2920 in 6. In 23F, ST242 was found in 5 isolates and ST81 in 4.
According to the Clinical and Laboratory Standards Institute standards, overall 38% of the isolates were penicillin nonsusceptible (Table, Supplemental Digital Content 1, http://links.lww.com/INF/A344). In serotype 3, 6A and 6B, all the STs were susceptible to penicillin, but susceptibility was low (<15%) in major STs of 14 (ST13 and ST876) and 19A (ST320). Among ST320, 2 isolates were penicillin-susceptible, 6 were resistant and eleven were intermediately resistant. Among all serotypes, nonsusceptibility to ceftriaxone, imipenem and erythromycin was 36% (34/95), 71% (67/95), and 94% (89/95), respectively (Table, Supplemental Digital Content 1, http://links.lww.com/INF/A344).
By multilocus sequence typing analysis, we confirmed the continued prevalence of a few global STs since mid 1990s in Taiwan, including ST258 in 6A, ST90 and ST76 in 6B, and ST81 and ST242 in 23F.5 In addition, ST902 and ST320 were the 2 emerging STs found in our study. ST902 was first isolated in Singapore and recently in Taiwan in serotype 6B isolates only.6 However, in our study, ST902 isolates were more in serotype 6A than in 6B. Emergence and spread of ST902 in serotypes 6A and 6B and ST1569 in serotypes 3 and 14 in this region might be due to capsular switching between serotypes. Analysis of allelic profiles query results revealed that single locus variants of ST902 can only be found in East-Asian countries (available at: http://spneumoniae.mlst.net).
In serotype 19A, ST320 was predominant. None of the previous studies reported the presence of the globally spreading ST320 in Taiwan and our data indicate that the highly drug resistant isolates of ST320 emerged in 2007 and has become prevalent in Taiwan since then.3,5,6 Nine of the 19 isolates in serotypes 19A were multidrug-resistant. Clonal expansion of serotype 19A in many countries is being investigated widely.7,8,12,13 In United States where PCV7 is widely used, the incidence of pneumococcal diseases caused by serotype 19A has been increasing.13 Among the penicillin-resistant 19A isolates clonal complexes CC199 and CC320 expanded rapidly after PCV7 introduction, and ST320 is a predominant sequence type in CC320. ST320 was not detected in surveillance studies before PCV7 introduction in United States and Spain.8,13 Recently, Choi et al, reported expansion of multidrug-resistant serotype 19A clone in Korean children before the introduction of PCV7 and suggested that the clonal expansion of ST320 was responsible for the increase of serotype 19A isolates. In addition, all 19A isolates from 2002 to 2006 in Korean hospital were of ST320.12 Because the PCV7 was not available in Taiwan until October 2005, further studies are needed to examine whether the clonal expansion is linked to the partial introduction of PCV7 in Taiwan.6 Emergence of penicillin-nonsusceptible ST320 similar to the Korean study also indicates that the clonal expansion might be related to antibiotic overuse in Taiwan as well as in Korea. From this perspective, since ST320 is a double locus variant (DLV) of ST236, emergence of ST320 from resistant serotype 19F might be due to 2 events, capsular switching and mutation in 2 loci. This is possible because in the present study ST236 and its variant ST2920 were the 2 major STs found in serotype 19F isolates, and both single locus variants and double locus variants of ST236 have been reported in many geographically distinct areas such as Greece, New Zealand, United States, and Taiwan.1,2,4
In CGCH the penicillin resistance rate is 6.3%, which is similar to the current resistance rate reported in Taiwan.6,14 Among the serotype 3 isolates, ST180 remains the predominant ST and all the isolates were susceptible to penicillin. Globally serotype 3 isolates remain susceptible to many antibiotics probably due to low competence.1,15 Although Hsieh et al found that the isolates sensitive to penicillin were significantly less competent,15 we found 100% susceptibility to penicillin in highly competent 6B isolates, suggesting that in addition to competence other mechanisms (genotype, mutations, capsule, drug pressure, etc.) are required for a serotype to acquire resistance to penicillin. On the other hand, presence of highly invasive potential in serotypes 14 and 19A might be associated with increasing antimicrobial resistance.3,16 Analysis of the recently sequenced genome of the invasive serotype 14 revealed that the serotype obtained new virulence genes after adaptations to an environment with high drug pressure by laterally acquiring many mobile genetic elements carrying 8 antimicrobial resistance genes.16
In regions like Taiwan with a high antibiotic selective pressure and suboptimal vaccine coverage, global resistant clones continue to circulate and new clones with higher resistance and invasive potential are emerging. To best prevent IPD as well as the dissemination of resistance, universal rather than optional use of PCV7 or the next-generation broad range PCV (PCV10 or PCV13) should be adopted.
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