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.

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]-[12]. In 2005, two predominant macrolide-resistant S. pneumoniae CCs, namely CC271 and CC15, were identified in New South Wales, Australia [13]. Recently, Tsai et al. reported the prevalence of serotype 19A pneumococcal isolates increased significantly in Taiwan from 2006 to 2010 and that more than 90% of the isolates were non-susceptible to azithromycin [14]. In the current study, we investigated the molecular characteristics of azithromycin-resistant S. pneumoniae recovered from various clinical sources of patients who were treated at 22 different hospitals in Taiwan from 2006 to 2010.

Methods

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].

Multilocus sequence typing (MLST)

MLST was performed as described previously [19]. Allele profiles and sequence types were determined using the MLST database (http://spneumoniae.mlst.net/).

Statistical analysis

Statistical analyses were conducted using GraphPad Prism V5.0 (GraphPad Software, San Diego, CA, USA).

Results

Prevalence of isolates carrying the erm(B) and mef(A)genes

Among the 486 isolates, 59% carried the erm(B) gene, 22% carried the mef(A) gene, and 19% carried both genes (Table 1). The prevalence of isolates carrying the erm(B) gene did not differ significantly from year to year (p-value = 0.2436) (Table 1); the prevalence of isolates carrying the mef(A) gene declined significantly from 30% in 2006 to 5% in 2010 (p-value = 0.0001); and the prevalence of isolates carrying both genes increased significantly from 10% in 2006 to 25% in 2010 (p-value = 0.0136) (Table 1). There were no obvious geographic differences with respect to the distribution of isolates carrying erm(B), mef(A), or both genes (data not shown).

Table 1

Prevalence of and genes among azithromycin-resistant isolates from 22 hospitals in Taiwan from 2006 to 2010

Resistant gene	No. (%) of isolates, by study period					Total (n = 486)
	2006	2007	2008	2009	2010
	(n = 110)	(n = 110)	(n = 153)	(n = 53)	(n = 60)
erm(B)-positive	66 (60)	65 (59)	92 (60)	24 (45)	42 (70)	289 (59)
mef(A)-positive	33 (30)	26 (24)	33 (22)	11 (21)	3 (5)	106 (22)
mef(A)/erm(B)-positive	11 (10)	19 (17)	28 (18)	18 (34)	15 (25)	91 (19)
P-value*	-	0.1683	0.8716	0.0224	0.3091

*P-value for temporal change of mef(A)/erm(B)-positive by study period.

Serotype and sequence type of isolates carrying both erm(B) and mef(A) genes

All isolates of serotype 3 and 15B carried only erm(B) (Table 2). The majority of the other main serotypes also carried only the erm(B) gene, namely serotype 23 F (73%), 14 (87%), 23A (86%), and 6B (54%) (Table 2). Among serotype 19 F isolates, 13% carried the erm(B) gene, 36% carried the mef(A) gene, and 52% carried both genes. The majority of serotype 19A isolates carried both erm(B) and mef(A) genes (61%) (Table 2). Of 91 these isolates carried both genes, 58 (64%) of isolates were belong to serotype 19 F, followed by 19A (22/91; 24%).

Table 2

Correlation between the main serotypes of azithromycin-resistant isolates and macrolide-resistant genes

Serotype*	No. of isolates	No. (%) of isolates, by resistant gene
		erm(B) -positive	mef(A) -positive	mef(A)/erm(B) -positive
19F	112	14 (13)	40 (36)	58 (52)
23 F	90	66 (73)	21 (23)	3 (3)
14	71	62 (87)	8 (11)	1 (1)
6B	66	35 (53)	29 (44)	2 (3)
19A	36	10 (28)	4 (11)	22 (61)
3	23	23 (100)	0 (0)	0 (0)
15B	15	15 (100)	0 (0)	0 (0)
23A	16	14 (88)	1 (6)	1 (6)
NT	28	25 (89)	3 (11)	0 (0)
Others**	29	25 (86)	0 (0)	4 (14)

NT: nontypeable.

*Data of serotype were used in this study as reported by Tsai HY et al. [14],

**Include 9 V (n = 9), 6A (n = 9), 10A (n = 2), 20 (n = 1), 11A (n = 1), 15A (n = 5), 22 F (n = 2).

The distribution of isolates harboring erm(B) and mef(A) genes by sequence type was 33% for ST320, 32% for ST236, 12% for ST271, 8% for ST81, 2% for ST283 and ST8525, and 11% for other sequence types (Table 3). The majority of ST236 (28/29) and ST271 (10/11) clones belonged to serotype 19 F. Isolates of clone ST320 mainly belonged to serotype 19 F (16/30) and serotype 19A (13/30) (Table 3). Seven isolates of clone ST81 were identified as belonging to serotype 23 F (n = 2), 23A (n = 1) and 6A (n = 4). Based on the results of MLST allelic profiling, the ST236 and ST81 clones were identified as reference strain of PMEN global clone Taiwan19F-14 and reference strain of PMEN global clone Spain23F-1 respectively (Table 3). ST320 and ST271 clones were identified as a double-locus variant (DLV) and a single-locus variant (SLV) of the worldwide-established Taiwan19F-14 (ST236) clone respectively.

Table 3

Sequence type and Serotype of azithromycin-resistant isolates with PCR positive for / genes

ST	No. (%) of isolates	Serotype (no. of isolates)	Related PMEN clone [ [20]] **
236	29 (32)	19 F (28), 19A (1)	Taiwan19F-14/ST236
320	30 (33)	19F (16), 19A (13), 14 (1)	DLV of Taiwan19F-14/ST236
271	11 (12)	19 F (10), 19A (1)	SLV of Taiwan19F-14/ST236
81	7 (8)	23 F (2), 23A (1), 6A (4)	Spain23F-1/ST81
283	2 (2)	19 F (2)	-
8525	2 (2)	19 F (2)	-
Others*	10 (11)	19 F (4), 19A (3), 23 F (1), 6B (2)	-

ST: Sequence type; PMEN: Pneumococcal molecular epidemiology network.

*Others (n): ST3111 (1), ST3164 (1), ST6993 (1), ST7123 (1), ST237 (1), ST257 (1), ST3625 (1), ST2993 (1), ST76 (1), new ST (1); DLV, double locus variant; SLV, single-locus variant;

**PMEN website. Available: http://web1.sph.emory.edu/PMEN/pmen_table2.html.

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%).

Figure 1

A phylogenetic tree analysis based on pulsed-field gel electrophoresis profiles with I among isolates of azithromycin-resistant carrying both genes.

Table 4

Pulsed-field gel electrophoresis (PFGE) clusters, serotypes and sequence types of azithromycin-resistant isolates with PCR positive for / positive genes

PFGE cluster*	No. of isolates (n = 87)**	Serotype (no. of isolates)	Sequence type (no. of isolates)
I	4	19 F (3), 19A (1)	320 (2), 236 (2)
II	16	19 F (12), 19A (4)	320 (9), 236 (2), 271 (5)
III	7	19 F (7)	236 (6), 271 (1)
IV	3	19 F (3)	320 (1), 236 (1), 237 (1)
V	9	19 F (9)	236 (9),
VI	24	19 F (11), 19A (13)	320 (13), 236 (3), 271 (3), 6993 (1) 3164 (1), 7123 (1), 8525 (1), 283 (1),
VII	4	19 F (4)	320 (1), 236 (1), 271 (1), 283 (1)
VIII	5	19 F (1), 6A (2), 23 F (1), 23A (1),	236 (1), 81 (4)
Un-clustered	15	19 F (7), 19A (3), 23 F (2), 6B (1), 6A (1), 14 (1)	320 (3), 236 (4), 271 (1), 81 (2), 76 (1), 275 (1), 2993 (1), 3111 (1), 3625 (1)

*PFGE-based clusters were defined as groups of 3 or more isolates with ≥80% similarity on the dendrogram.

**PFGE data were not available for four isolates (H03-020-2010, H04-004-2008, H12-017-2009, H15-012-2008).

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 pneumococcal conjugate vaccine in Taiwan. These findings are in line with a previously published report on the PROTEKT US surveillance study from 10% in 2000 1 to 16% in 2003 [21], the study in Alaska from 0% in 1986 to 21% in 2010 [8], and the study in Canada from 3% in 1998 to 19% in 2008 [22].

In this study, the majority of azithromycin-resistant isolates carrying both mef(A) and erm(B) genes was serotype 19 F (58/91; 64%), followed by19A (22/91; 24%) and is similar to a previous published report in Korea, 57% of carried both genes were serotype 19 F (44/77, 57%), followed by 19A (21/77, 30%)7.5%) [23]. However, the study in Alaska showed 79% of isolates carrying both genes was serotype 19A (15/19), followed by 19 F (3/19; 16%) [8].

We investigated further via MLST and PFGE all isolates carried both the erm(B) and mef(A), and identified 33% of these to be of ST320, followed by ST236 (32%), ST271 (12%), and other STs (23%). Previously, it was reported in Taiwan that the CCs related to Spain23F-1, Taiwan19F-14, and Taiwan23F-15 were responsible for the spread of isolates with high-beta-lactam resistance [24],[25]. Recently, the S. pneumoniae serotype 19A ST320 clone, derived from an international Taiwan19F-14 (ST236) clone, has become prevalent in many countries, including Taiwan [25]. In Arizona, the isolates carrying both mef(E)/erm(B)-positive genes are multidrug-resistant clonal lineages of Taiwan19F-14 [9].

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 time- and 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.