Increase in the rate of azithromycin-resistant Streptococcus pneumoniae isolates carrying the erm(B) and mef(A) genes in Taiwan, 2006–2010

Background This study investigated the molecular characteristics of azithromycin-resistant Streptococcus pneumoniae in Taiwan. Methods A total of 486 non-duplicate isolates of azithromycin-resistant S. pneumoniae recovered from various clinical sources of patients treated at 22 different hospitals in Taiwan from 2006 to 2010. The presence of erm(B) and mef(A) genes using duplex PCR, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis of these isolates were studied. Results Of the isolates tested, 59% carried the erm(B) gene, 22% carried the mef(A) gene, and 19% carried both genes. The prevalence of isolates carrying the erm(B) and mef(A) genes increased from 10% (11/110) in 2006 to 25% (15/60) in 2010 (p-value = 0.0136). The majority of isolates carrying both erm(B) and mef(A) genes belonged to serotypes 19 F (64%) followed by 19 F A (24%). Of these isolates, 33% were sequence type 320 (ST320), 32% were ST236, and 12% were ST271. Conclusions The increase in incidence of mef(A)/erm(B)-positive azithromycin-resistant S. pneumoniae isolates during the study period was primarily due to serotypes 19 F and 19A and ST236 and ST320. Electronic supplementary material The online version of this article (doi:10.1186/s12879-014-0704-z) contains supplementary material, which is available to authorized users.


Background
Streptococcus pneumoniae is a leading cause of bacterial pneumonia, meningitis, and sepsis worldwide. Since 1965, many cases of infections due to drug-resistant S. pneumoniae have been reported [1]. The emergence of antimicrobial resistance is correlated with selective pressure from the use, often inappropriate, of antimicrobial agents and results in increased mortality, morbidity, and health care costs [2]. Antibacterial resistance in S. pneumoniae is increasing, affecting principally β-lactams and macrolides (azithromycin, erythromycin, or clarithromycin) with prevalence ranging between 1% and 90% depending on the geographical area [3]. Fluoroquinolone resistance has also been reported in countries with high levels of antibacterial resistance and consumption [3].
Macrolide resistance in S. pneumoniae is most often mediated by two mechanisms: target-site modification encoded by the erm(B) gene and active drug efflux mediated by a membrane efflux pump encoded by mef-class genes [4]. Song et al. reported the erm(B) gene was found in >50% of pneumococcal isolates either alone or in combination with mef(A) among S. pneumoniae isolates from 10 Asian countries during 1998 2001 [5]. In Finland and Germany, the most frequent macrolide resistance determinant carried was the mef gene [6,7]. Macrolide resistance among pneumococcal isolates in Alaska recovered from 1986 2010 was also reported to be predominantly mediated by mef genes and this has not changed significantly over time [8]. However, the authors of the study reported a significant increase in the proportion of isolates that possess both erm(B) and mef(A), primarily among serotype 19A isolates.
Bowers et al. reported that of 592 clinical pneumococcal isolates collected in Arizona from 1999 to 2008, all isolates carrying the erythromycin-resistant genes mef(E) and erm(B) were multidrug-resistant clonal lineages of Taiwan 19 F-14 and most were multilocus sequence type (ST) 320 [9]. In China, recent studies have shown that erythromycin-resistant isolates commonly carry both genes and that the majority of isolates belong to ST271, ST320, ST236, with clonal complex 271 (CC271) being the most frequently isolated CC [10][11][12]

Bacterial isolates
A total of 530 consecutive and non-duplicate pneumococcal isolates were collected from various clinical specimens of patients treated at 22 different hospitals in Taiwan during a 3-month period per year, with a maximum number of isolates per year of 10 during 2006 2008 and 5 during 2009 2010 [14]. Among these isolates, 486 were not susceptible to azithromycin [14]. These pneumococcal isolates were collected as part of the Tigecycline In Vitro Surveillance in Taiwan (TIST) study, a nationwide, multicenter, prospective surveillance study conducted in 12 regional hospitals (500 1000 beds) and 10 medical centers (1200 3000 beds) (eight in northern, four in central, six in southern and two in eastern Taiwan) from January 2006 to December 2010 [15], Pneumococcal isolates were identified at each hospital and the identification was confirmed by the central laboratory at the National Taiwan University Hospital [15]. Serotype determination by a latex agglutination method and antimicrobial susceptibility testing by the broth microdilution method were performed as described previously [14]. Isolates were collected as part of standard patient care and no ethical approval required for your use.

Detection of erm(B) and mef(A) genes
The detection of erm(B) and mef(A) was performed by duplex PCR as previously described [5,16].
Pulsed-field gel electrophoresis (PFGE) analysis PFGE analysis of isolates was performed as described previously [17,18]. The Dice coefficient of similarity was calculated and the unweighted pair group method with arithmetic averages (UPGMA) was used for cluster analysis. Isolates with coefficients of similarity ≥80% were considered to be the same cluster [18].

Clusters of isolates carrying both erm(B) and mef(A) genes
We constructed a phylogenetic tree based on PFGE profiles and found no specific clustering for the strains of serotype 19 F and 19A or for the three major sequence types (ST320, ST271, and ST236) ( Figure 1). In this study, the isolates carrying both erm(B) and mef(A) were stratified into eight clusters (Cluster I to VIII) by PFGE ( Figure 1 and Table 4). Clusters III, IV, V, and VII corresponded to the isolates with serotype 19 F (Table 4). Isolates belonging to the same cluster can have different serotypes and STs. Furthermore, several isolates with the same ST also exhibited different serotypes and pulsotypes. The majority of isolates of serotype 19A were in cluster VI (13/24, 54%). ST320 clone isolates belonged to cluster VI (13/24, 54%), cluster II (9/16, 56%), and cluster I (2/4, 50%). Meanwhile, isolates of clone ST236 were frequently clustered in cluster V (9/9, 100%) and III (6/7, 86%) ( Table 4). The majority of ST81 clone isolates were clustered in cluster VIII (4/5, 80%).

Discussion
Azithromycin is the most commonly used macrolide in the treatment of community-acquired pneumonia and other respiratory tract infections in Taiwan. The rate of susceptibility to azithromycin remained stationary from 2006 to 2010 in Taiwan, although the numbers of isolates randomly collected in 2009 and 2010 were lower than in 2006 to 2008 [14]. In Taiwan, PCV-7 vaccination was introduced in October 2005 and PCV-13 was introduced in July 2010. Nevertheless, some studies have shown that changes in antimicrobial susceptibility before and after implementation of the PCV-7 vaccine were not associated with serotypes [14]. Our finding of increase in the rate of azithromycin-resistant S. pneumoniae isolates carrying the erm(B) and mef(A) genes from from 10% in 2006 to 25% in 2010 after the introduction of the , **Include 9 V (n = 9), 6A (n = 9), 10A (n = 2), 20 (n = 1), 11A (n = 1), 15A (n = 5), 22 F (n = 2).    3% in 1998 to 19% in 2008 [22].
In the last two decades, PFGE and MLST have become the main genotyping methods for assessing the genetic diversity of isolates [26]. Although both methods are timeand labor-consuming, they are useful for studying the local and global epidemiology of S. pneumoniae. In the present study, discrepancies of typing results by these two methods occurred. Since pneumococci are capable of undergoing capsular switching and are recognized as one of the most recombinogenic bacteria, additional typing methods, i.e. multiple-locus variable number tandem repeat analysis and MILST, have been developed recently to offer better discrimination in S. pneumoniae isolates [26].

Conclusions
The increase in incidence of mef(A)and erm(B)-positive azithromycin-resistant S. pneumoniae isolates during the study period was primarily due to serotypes 19 F and 19A and ST236 and ST320.

Competing interests
The authors declare that they have no competing interest. This search was supported by a fellowship grant from the International Society of Chemotherapy Infection and Cancer (ISC).
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