National Surveillance on Vancomycin-Resistant Enterococcus faecium in Taiwan: Emergence and Widespread of ST414 and a Tn1546-Like Element with Simultaneous Insertion of IS1251-like and IS1678

Cases of bacteremia caused by vancomycin-resistant E. faecium (VRE-fm) increased significantly in Taiwan. The present multicenter surveillance study was performed to reveal the associated epidemiological characteristics. In 2012, 134 non-repetitive VRE-fm isolates were prospectively collected from 12 hospitals in Taiwan. Antimicrobial susceptibility, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and analysis of van genes and Tn1546 structures were investigated. Two isolates carried vanB genes, while all the remaining isolates carried vanA genes. Three isolates demonstrated a specific vanA genotype - vanB phenotype. Nine (6.7%) isolates demonstrated tigecycline resistance, and all were susceptible to daptomycin and linezolid. Molecular typing revealed 58 pulsotypes and 13 sequence types (STs), all belonged to three major lineages 17, 18, and 78. The most frequent STs were ST17 (n = 48, 35.8%), ST414 (n = 22, 16.4%), and ST78 (n = 16, 11.9%). Among the vanA harboring isolates, eight structure types of the Tn1546-like element were demonstrated. Type I (a partial deletion in the orf1 and insertion of IS1251-like between the vanS - vanH genes) and Type II (Type I with an additional insertion of IS1678 between orf2 - vanS genes) were the most predominant, consisted of 60 (45.5%) and 62 (47.0%) isolates, respectively. The increase of VRE-fm bacteremia in Taiwan may be associated with the inter- and intra-hospital spread of some major STs and horizontal transfer of vanA genes mostly carried on two efficient Tn1546-like elements. The prevailing ST414 and widespread of the Type II Tn1546-like elements are an emerging problem that requires continuous monitoring.


Introduction
Enterococci are a part of the normal intestinal flora in human and animals. They may also colonize many body sites of healthy individuals and cause opportunistic infection in immunocompromised patients. These organisms are also a common cause of hospital-acquired infection (HAI) and are associated with a substantial proportion of bloodstream and urinary tract infections. Enterococci are intrinsically resistant to a variety of antibiotics. Vancomycin is usually required for treatment, especially for invasive infections. Therefore, treatment of infections caused by vancomycin-resistant enterococci (VRE) may be difficult.
In Taiwan, VRE was firstly reported in 1996 [13]. According to the reports from the Centers for Disease Control, Taiwan, and others from Taiwan, a significant increase of VRE-fm infection has been noted since 2008, especially in the northern and central Taiwan [14][15][16]. High selective pressure from antimicrobial usage was found to have contributed to the increase of VRE-fm infections [17][18][19]. In 2012, a nationwide, multicenter surveillance study was prospectively conducted at 12 hospitals in Taiwan. The genotypes (pulsotypes, sequence types) of VRE-fm clinical isolates and van gene analysis, including structure analysis of vanA/ Tn1546, were investigated and reported herein.

Ethics statement
The present study aimed to characterize vancomycin-resistant E. faecium isolates using molecular methods. All isolates studied were prospectively collected from 12 hospitals in Taiwan. Clinical information of the patients was not required in this study. Because all microbial cultures were ordered by physicians due to clinical necessity and none was collected purposely for this study, patients' informed consents were not required and therefore were not collected.

Hospital settings and bacteria
Between January and December 2012, a total of 134 non-repetitive VRE-fm blood isolates were collected prospectively from 12 hospitals in Taiwan. A total of 9 medical centers (each consisted of 1000-4700 beds, respectively) and 3 regional hospitals (each with 500-1100 beds, respectively) distributed among 4 geographic regions of Taiwan were included in the study. These included the Chang Gung

Susceptibility testing
Antibiotic susceptibility testing of ampicillin, ciprofloxacin and high-level gentamicin (Becton Dickinson, Franklin Lakes, NJ, USA) was performed by a disc diffusion method. Minimum inhibitory concentrations (MICs) of vancomycin, teicoplanin, linezolid, daptomycin and tigecycline were evaluated with E-test strips (AB Biodisk, Solna, Sweden). The results were interpreted according to the recommendations by the Clinical and Laboratory Standards Institute [20]. The susceptibility for tigecycline was defined based on the European Committee on Antimicrobial Susceptibility Testing criteria (MIC#0.25 mg/mL) [21]. Staphylococcus aureus ATCC29213 and Enterococcus faecalis ATCC29212 were used as control strains.

Detection of the vancomycin resistance genes
DNA of the isolates was extracted by using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). The vancomycin resistance genes, vanA, vanB, vanC1 and vanC2/C3, were detected using a multiplex PCR as previously described [22].
Multilocus sequencing type (MLST) and pulsed-field gel electrophoresis (PFGE) MLST was performed according the method described previously [23]. The amplicons were purified using Microcon PCR Centrifugal Filter Devices (Millipore Corporation) and sequenced using an ABI 3100-Avant Genetic Analyzer (Applied Biosystems). The sequence type (ST) was determined through the comparison with the MLST database on the public domain (http://efaecium. mlst.net). Clustering analysis of the STs was performed by the eBURST program through the web site. PFGE was performed by a previously described method [24]. The resulting patterns were analyzed by BioNumerics (version 6.5, Applied Maths, Austin, Texas). PFGE patterns with more than 80% similarity were considered as closely related and categorized into the same pulsotypes.

Molecular analysis of the Tn1546-like elements
The presence and the configuration of the Tn1546-like elements were analyzed using a published PCR overlapping technique with 10 pairs of primers [25]. The Tn1546 prototype (GenBank accession no. M97297) was used as the reference. Amplicons with unexpected fragment sizes were subjected to further sequence analysis.

Statistical analysis
Statistical analysis was performed by the Chi-square test or Fisher's extract test when appropriated. A value of p,0.05 was considered statistically significant.

Antimicrobial susceptibility and van gene analysis
A total of 134 VRE-fm isolates were confirmed and enrolled in the present study. The majority (n5132, 98.5%) of the isolates carried a vanA gene. Vancomycin MICs were high (.256 mg/mL) among the isolates with the vanA genes, but various MICs (4 -.256 mg/mL) were observed for teicoplanin. Among them, 3 isolates demonstrated a specific vanA genotype-vanB phenotype [26] with the teicoplanin MICs ranged between 4-8 mg/mL. Another 2 isolates carried the vanB gene were associated with a much lower MICs (vancomycin, 8 mg/mL; teicoplanin, 1 mg/mL).

Characterization of Tn1546-like elements
Genetic structures of the Tn1546-like elements were further analyzed among the 132 vanA-carried VRE-fm isolates. Eight different Tn1546 structure types were detected among the VRE isolates studied herein (Fig. 3). The prototype of Tn1546 (GenBank accession no. M97297) was not found in any of the isolates studied. None of the isolates produced positive results by using the first PCR primer pair (P1-P2), while all isolates produced amplicons of the expected size when the 7 th primer pair (P13-P14) was used. Diverse results were found with the use of the other 8 primer pairs, and accordingly 8 structure types were identified.
Types I and II were the most predominant, consisted of 60 (45.5%) and 62 (47.0%) isolates, respectively. Type I was characterized by the insertion of an IS1251-like element at nt 5820, the intergenic region between the vanS and vanH genes. DNA sequences of the IS1251-like element were identical to those published in the GenBank database with accession number AF148130. Eight STs (ST17, ST18, ST64, ST78, ST203, ST262, ST414, ST767) were found in Type I. Type II was similar to Type I except that an additional insertion of IS1678 at nt 3896, the intergenic region of orf2-vanS genes, was noted. One more difference was found in one Type II isolate in which the insertion of IS1251-like was found at nt 5812. Type II also consisted of 8 STs: ST17, ST78, ST203, ST252, ST262, ST323, ST341 and ST414. Among them, 4 STs were solely associated with one single structure type of Tn1546: ST18, ST64 and ST767 were found only in Type I, while ST341 was only in Type II.The remaining 6 structure types were sporadically found in 1-3 isolates, respectively (Fig. 3). The insertion of IS1251-like found in Types I and II were also found in Types III,VI, making the insertion of IS1251like the most predominant insertion sequences (128 isolates, 97.0%) among the isolates studied. The extra insertion of IS1678 found in Type II also could be found in Types III and VI (65 isolates, 49.2%). Large truncations in the regions consisting of orf1, orf2 and vanR and/or those consisting of vanX,vanY, or vanZ were found in Types IV, VII and VIII, as no amplicons could be obtained by the associated primer pairs.
The association of various structure types of Tn1546 with the major pulsotypes A-G was different. Pulsotypes A, B, and G were all associated with Type I, while Type II was found in all pulsotypes D and E isolates and prevalent in pulsotypes C and F. In pulsotype F, the five isolates from the northern region all carried the Type II Tn1546. Accordingly, among the 5 genotype clusters found in hospitals C 1 , N 1 and N 2 , ST17/pulsotype A, ST17/pulsotype B, and ST18/pulsotype G were all associated with Type I, while ST341/pulsotype D and ST414/pulsotype E were associated with Type II.

Discussion
A total of 13 STs were identified in the present study. All of them belonged to CC17, the most frequently identified clonal complex globally [28]. CC17 is characterized by the multi-drug resistance to ampicillin and fluoroquinolones and is a globally distributed nosocomial-related lineage [9,29]. Among them, three STs, ST17, ST414 and ST78, were predominant during the study period and could be identified from different regions in Taiwan. ST17 and ST78 were the worldwide spreading epidemic clones, and have been identified among hospitalized patients in almost all continents, including Asia, North and South America, Europe, and Australia [11,[30][31][32][33]. In contrast, ST414 (16.4%) was the second most prevalent ST in the present study but was less common in other countries. In fact, it was firstly identified from Australia in 2008 [34]. Later, recent reports from Asia, including China [6,35], Hong Kong [36], and Taiwan [15,19], also demonstrated the emergence of ST414. According to a recent outbreak investigation from northern Taiwan, the proportion of ST414 could be as high as close to 50% [19]. However, up to the present, ST414 has not yet been reported from the western countries. Whether or not this newly emergent Asia clone, ST414, will soon be discovered or widespread in western countries warrants close monitoring. Vancomycin resistance is due to the presence of van genes, including vanA, vanB, and other minor van genes [10]. In the present study, all but two isolates carried the vanA gene. The results were consistent with reports from other countries [4,37]. However, the proportions of vanB-containing isolates in these studies were about 10%-20%, much higher than the ,2% found in the present study. The results suggested that the increase of VRE-fm isolates in Taiwan may be related to the spread of the vanA gene. Furthermore, previous reports from Taiwan demonstrated a high prevalence (51%-90%) of VRE isolates showing a specific VanB phenotype-vanA genotype [7,14,22]. In the present study, however, the proportion of such isolates greatly reduced to 2.3%. Reports from other countries remained rather constant at 5%-12% [6,33]. Therefore, the decrease of such specific vanA genotype -VanB phenotype among the VRE isolates in Taiwan as presented in this study remains to be elucidated.
The vanA gene is known to be carried by Tn1546 [25]. Several genetic arrangements of the Tn1546 have been reported [8,11,12]. In the present study, we also identified eight structure types of Tn1546, with Types I and II being the most prevalent. Type I is characterized by the insertion of IS1251-like, which could also be found in Types II-VI. Actually, only four (3.0%) of our study isolates did not have this insertion. Insertion of IS1251 in the Tn1546 was firstly described in the United States in 1995 [38]. In 1999, Willems et al. reported a Tn1546 F1 Type [12], in which the IS1251 was also inserted between vanS and vanH genes, similar to our Type I structure. Recent reports indicated that such a structure was not only prevalent in Taiwan (36.7%), but also in Brazil (87.8%), and Paraguay (92.5%) [11,30,32]. Type II is characterized by an extra insertion of IS1678 between orf2-vanS genes. IS1678 was only recently reported in Korea with a different insertion site at the intergenic region between vanX and vanY genes [39]. Therefore, the finding that almost a half of the VRE-fm isolates studied herein carried a Tn1546 of this structure type is surprising. Moreover, according to our preliminary data on the 152 VRE isolates collected in 2013, the proportion of the isolates carrying this Type II Tn1546 has increased to ,60%. The mechanism underlying the emerging and widespread of the Type II Tn1546 warrants further studies.
Another characteristic of the present study was that the left extremity of Tn1546 was lost in all isolates studied. Several reports also demonstrated similar findings [11,12,30,32,40]. This region corresponded to gene orf1 which was associated with transposition functions [10]. A previous study indicated that this truncated Tn1546-like element plus the insertion of IS1251-like was associated with a much higher conjugation frequency compared to that shown by the prototype Tn1546 (10 26 vs. 7610 27 ) [11]. It seems that such a structural change (truncated orf1 plus insertion of IS1251-like) in Tn1546 may facilitate the dissemination of the transposon, so as that of the van gene cassette. It may also explain the finding that the majority (95.5%) of the isolates studied herein carried a Tn1546-like element that consisted of such a changed structure. The less dissemination ability of the prototype Tn1546 may lead to the decrease of its prevalence. Therefore, similar to a recent study [41], we could not find the prototype in any of our isolates studied.
In conclusion, our results revealed highly diverse genotypes among the VRE clinical isolates in Taiwan. However, we also found the predominance of three major ST types (ST17, ST414, ST78), seven predominant pulsotypes, and two prevailing structure types of the Tn1546-like element (Types I and II) from different hospitals and regions in Taiwan. Therefore, both the horizontal transfer of vanA genes through some efficient Tn1546-like elements and the clonal dissemination of some major genotypes within or among the hospitals may have contributed the overall increase of VRE in Taiwan. Furthermore, mechanisms underlying the high proportion of ST414 and isolates carrying the Type II Tn1546-like elements may warrant a further study.