Characteristic of Enterococcus faecium clinical isolates with quinupristin/dalfopristin resistance in China

Background Quinupristin/dalfopristin (Q/D) is a valuable alternative antibiotic to vancomycin for the treatment of multi-drug resistant Enterococcus faecium infections. However, resistance to Q/D in E. faecium clinical isolates and nosocomial dissemination of Q/D-resistant E. faecium have been reported in several countries and should be of concern. Results From January 2012 to December 2015, 911 E. faecium clinical isolates were isolated from various specimens of inpatients at the first Affiliated Hospital of Wenzhou Medical University located in Wenzhou, east China. Of 911 E. faecium clinical isolates, 9 (1.0 %, 9/911) were resistant to Q/D, with the Q/D MIC values of 64 mg/L(1), 32 mg/L(1), 16 mg/L(3), 8 mg/L(1) and 4 mg/L(3) determined by broth microdilution. All Q/D-resistant isolates were susceptible to vancomycin, tigecycline and teicoplanin but resistant to penicillin, ampicillin and erythromycin. vatE was only found in one Q/D-resistant E. faecium isolate while vatD was not detected in any of the isolates tested. 8 of 9 Q/D-resistant E. faecium isolates were found be positive for both ermB and msrC. The combinations of Q/D resistance determinants were ermB-msrC (7 isolates) and ermB-msrC-vatE (one isolate). ST78, ST761, ST94, ST21 and ST323 accounted for 4, 2, 1, 1 and 1 isolate, respectively, among which ST78 was the prevalent ST. Conclusion Q/D-resistant E. faecium clinical isolates were first described in China. Carriage of vatE, ermB and msrC was responsible for Q/D resistance.


Background
Enterococci, which are the normal commensals in the intestinal tract of humans and animals, are the common cause of nosocomial infections. Enterococcus faecalis and Enterococcus faecium are responsible for most of enterococcal infections, such as urinary-tract infections, intra-abdominal infections, bacteremia, and endocarditis [1]. With an intrinsic and acquired resistance to some antimicrobial agents, Enterococci have become important nosocomial pathogens [2]. Infections caused by multidrug -resistant Enterococci, especially multiple resistances to vancomycin, penicillin, and aminoglycoside (high-level resistance), are of a major concern, making enterococcal infections a serious and life-threatening disease [1]. Increase of vancomycin-resistant Enterococci (VRE) limits the selection of vancomycin for treatment of enterococcal infections. Therefore, attention has been directed toward the alternatives for the treatment of enterococcal infections, especially VRE infections.
Quinupristin/dalfopristin(Q/D) is a combination of streptogramins B (quinupristin) and streptogramins A (dalfopristin) at 30:70 ratio. Streptogramins A and B are bacteriostatic when used separately but act synergistically when combined. The synergic behavior of the combination results in Q/D rapidly bactericidal against the majority of Gram-positive organisms [3]. Clinically, Q/D is mainly used for the treatment of infections caused by multidrug-resistant Gram-positive cocci [4,5]. Q/D is effective against E. faecium, but not against E. faecalis [6].
As E. faecalis isolates possess a chromosomal gene named lsa responsible for lincosamide and streptogramin A resistance, which results in all E. faecalis with intrinsic resistance to Q/D [2]. Q/D has been successfully used for the treatment of vancomycin-resistant E. faecium (VREf) infections [7,8]. The combination of Q/D and high-dose ampicillin was used successfully for treating persistent bacteremia and endocarditis caused by VREf [5,9]. Q/D is a valuable alternative to vancomycin for the treatment of multi-drug resistant E. faecium infections. However, resistance to Q/D in E. faecium clinical isolates and nosocomial dissemination of Q/D-resistant E. faecium have been found in several countries [10][11][12][13]. Although the prevalence of Q/D resistance among these isolates from humans is still low, emergence and dissemination of Q/D-resistant E. faecium limit the therapeutic option of successful treatment of VREf infections. However, Q/D resistance among E. faecium isolates from inpatients has not been reported in China. The aim of the present study was to elucidate the prevalence of Q/D resistance among E. faecium isolates from the various specimens of inpatients at a tertiary teaching hospital between January 2012 and December 2015.

Collection of clinical isolates and E. faecium identification
From January 2012 to December 2015, a total of 911 non-duplicate E. faecium isolates (single isolate per patient) from various specimens of inpatients in the first Affiliated Hospital of Wenzhou Medical University located in Wenzhou, east China, were collected consecutively for investigating the prevalence of Q/D resistance. When multiple E. faecium isolates were isolated from the same patient, the first isolated strain was included. The included isolates were identified as E. faecium using Gram's stain, catalase test and Vitek-2 microbiology analyzer (bioMe'rieu, Marcy l'Etoile, France). Q/D-resistant isolates were re-identified as E. faecium using Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) (bioMe'rieux, Marcy l'Etoile, France) and PCR amplifying 16S rRNA gene. S. aureus ATCC25923, Escherichia coli ATCC25922 and Pseudomonas aeruginosa ATCC27853 were used as control strains for identification of bacteria. The Ethics Committee of the first Affiliated Hospital of Wenzhou Medical University exempted this study from review because the present study focused on bacteria.

Antimicrobial susceptibility testing
The antimicrobial susceptibility testing for E. faecium clinical isolates was determined by Vitek-2 microbiology analyzer (bioMe'rieu, Marcy l'Etoile, France) in accordance with the manufactory's instructions. Q/D resistance initially determined by Vitek-2 microbiology analyzer was reconfirmed using disk diffusion method were according to the guidelines provided by the Clinical and Laboratory Standards Institute (CLSI) [14]. Q/D minimum inhibitory concentration (MIC) values for Q/Dresistant E. faecium clinical isolates were determined twice using broth microdilution method operated by two separate operators in accordance with the guidelines recommended by CLSI [14]. Q/D (Synercid, Astellas Pharma, Inc.) was kindly provided by Pro. Nobumichi Kobayashi from Sapporo Medical University (Japan). S. aureus ATCC25923, E. coli ATCC25922 and P. aeruginosa ATCC27853 were used as control strains for antimicrobial susceptibility testing.

Detection of determinants responsible for Q/D resistance
The genes responsible for resistance to streptogramin B antibiotics including erm (ermA, ermB and ermC) and msrC, and streptogramin A antibiotics including vat (vatD and vatE) were detected by PCR assays with specific primers and reaction conditions described previously [15][16][17]. DNA sequencing was used for the identification of genotype of the genes tested.

Multi-locus sequence typing (MLST)
MLST of E. faecium isolates was performed using amplification of internal fragments of the seven housekeeping genes including adk , atpA , ddl , gyd, gdh, purK and pstS of this organism as described previously [18]. Following purification and sequencing of these genes, the sequences were compared with the existing sequences available on the MLST website for E. faecium (http://efaecium.mlst.net), and STs were determined according to the allelic profiles.

Results and discussion
Prevalence of Q/D resistance among E. faecium clinical isolates Among 911 E. faecium clinical isolates over the study period, 9 (1.0 %, 9/911) were resistant to Q/D determined by the Vitek-2 Automated Microbiology Analyzer with GPS card and disk diffusion method. The Q/D MIC values for 9 Q/D-resistant E. faecium clinical isolates determined by broth microdilution method were as follows: 64 mg/L, 1; 32 mg/L, 1; 16 mg/L, 3; 8 mg/L, 1 and 4 mg/L, 3 ( Table 1). All Q/D-resistant isolates were susceptible to vancomycin, tigecycline and teicoplanin but resistant to penicillin, ampicillin and erythromycin. Eight (88.9 %) of 9 isolates with Q/D resistance were resistant to moxiofloxacin, levofloxacin and ciprofloxacin. Five, 1 and 5 isolates were resistant to gentamicin (high-level resistance), linezolid and nitrofurantoin, respectively ( Table 1). As Q/D is not available in China, the patients infected by these Q/D-resistant E. faecium isolates were not subject to be treated by Q/D. Therefore, we speculate that the acquisition of Q/D resistance among E. faecium clinical isolates in the present study was not associated with the consumption of Q/D. The previous study reported that the extensive in-feed use of virginiamycin showed full cross-resistance with Q/D selected for streptograminresistant enterococci and resulted in a reservoir of resistance genes in production animals [19]. Q/D resistance determinants from animal-associated enterococcal isolates can spread to E. faecium human isolates [20]. However, in the present study, it remains unknown whether these Q/D-resistant E. faecium clinical isolates was indeed associated with the isolates from animals. To our knowledge, the present study is the first report of Q/D resistance among E. faecium clinical isolates in China. Up to now, although the prevalence of Q/D resistance among E. faecium clinical isolates was very low, the intermediate resistance to Q/D was relatively high. A investigation from Japan reported that none was resistant to Q/D while 28 (17.6 %, 28/159) were intermediate resistant to this antimicrobial agent (MIC = 2 mg/L) among 159 E. faecium isolates from clinical specimens in a Japanese hospital from 1997 to 2006 [21]. A study from Greek reported that 250 of 865 (28.9 %) E. faecium isolates from patients of eight Greek hospitals between 2005 and2006 were intermediate-resistant to Q/D (MICs = 1.5-4 mg/L) [22]. In another report, all 60 primary clinical isolates of E. faecium with resistance to glycopeptides were fully susceptible to Q/D, with MIC50 and MIC90 values of 1.0 mg/L and 1.5 mg/L [23]. These previous studies and our study support the evidence that Q/D is still an effective and valuable antimicrobial agent for treating infections caused by multi-resistant E. faecium, even VREf. Nevertheless, the emergence of Q/D resistance, especially increased intermediate resistance to this antimicrobial agent, has become a concern. Moreover, a high prevalence (10.0 %) of Q/D resistance among E. faecium clinical isolates was found in Korea, which was associated with both clonal spread and the sporadic emergence of Q/D-resistant isolates [12]. Hsueh et al. also reported a similar high prevalence (9 %) of Q/D resistance among vancomycinresistant E. faefium clinical isolates in Taiwan [24]. As our study did not investigate the prevalence of intermediate resistance to Q/D among E. faecium clinical isolates, it remains unclear whether there was a trend of increase in intermediate resistance to Q/D, which should be further investigated.

Determinants of Q/D resistance among E. faecium clinical isolates
The resistance to Q/D is associated with enzymatic modification of the antibiotic, active transport or efflux mediated by an ATP-binding protein, and alteration of the target site [6]. Both resistance to streptogramin A and streptogramin B is necessary for occurrence of Q/D resistance [25]. Carriage of more than one streptogramin A resistance gene (vat or vga) is necessary for the occurrence of Q/D resistance in an organism [6]. The streptogramin A resistance genes found in E. faeciumare were vatD and vatE encoding acetyltransferases [6]. Modification of dalfopristin by the acetyltransferases VatD and VatE renders it ineffective, abolishing the synergy with quinupristin. vatD was initially found in an E. faecium isolate from a hospitalized patient in Europe [26].vatD and vatE have been found in E. faecium animal and human isolates in Europe, USA and Korea [6,27,28]. In contrast, some studies reported that none of the genes involved in the expression of dalfopristin resistance (vatD, vatE, vgaA and vgaB) were found in any Q/D-intermediate-resistant or -resistant E. faecium isolates [21,22,27]. Similarly, in the present study, vatE was only found in one Q/Dresistant E. faecium isolate while vatD was not detected in any of the isolates tested ( Table 1). The most commonly known resistance to streptogramin B in enterococci is the macrolides, lincosamides, and streptogramins B ( MLSB) resistance conferred by the erm genes (ermA, ermB and ermC) encoding an enzyme that dimethylates an adenine residue in the 23S rRNA, which results in decreased  Q/D resistance  determinants   1  pus  78 16  MOX, GEN(H), P, E, LZD, F, LEV, AMP, CIP TEC, TGC, TET,  binding of these antimicrobial agents [6]. In the present study, ermB was found among 8 of 9 Q/D-resistant E. faecium isolates. vgbA encoding a streptogramininactivating enzyme (lyase) has been found rarely in the isolates of E. faecium, but msrC conferring resistance to streptogramin B antibiotics by active transport is commonly found in E. faecium isolates [29]. In the present study, 8 of 9 Q/D-resistant E. faecium isolates were found be positive for msrC. The remaining one isolate was negative for all resistance genes tested. Currently, there has not been reported the combination of multiple streptogramin A resistance genes in E. faecium. However, combinations of the vatD-vgbA and ermB-vatD or vatE genes were found in Q/D-resistant isolates [10,30,31]. The vatE was more common than vatD in animal sources [11,28,32]. In the present study, the combinations of Q/D resistance determinants were ermB-msrC (7 isolates) and ermB-msrC-vatE (one isolate).

Molecular characteristics of Q/D-resistantE. Faecium clinical isolates
The lineage clonal complex 17 (CC17) (ST17, ST18, and ST78) significantly associated with hospital infections emerges as the high-risk clone responsible for the worldwide spread of VERf [33]. A report from Poland showed the domination of representatives of lineages ST78 and ST17/18 (52.7 and 46.4 %, respectively) among consecutive E. faecium clinical isolates collected in 30 hospitals between May 2010 and June 2011 through prospective surveillance in Poland [34]. VERf isolates from 10 infected and 40 colonized inpatients from a single hospital in the north of Spain were assigned to ST17 by MLST [35]. In China, ST78 was the predominant MLST type among VERf clinical isolates [36]. However, the molecular characteristic of Q/D-resistant E. faecium clinical isolates is limited. Among 25 Q/D-resistant E. faecium isolates from Korea, 10, 9 and 4 belonged to ST78, ST192 and ST203, respectively, with ST78 being the prevalent ST [12]. In the present study, among 9 Q/D-resistant isolates, ST78, 761, 94, 21 and 323 accounted for 4, 2, 1, 1 and 1 isolate, respectively, with ST78 being the prevalent ST (Table 1). In another report, 5 STs including four STs of CC17 were identified in Q/D-intermediate resistant E. faecium clinical isolates [21]. In addition, only a VERf isolate from China was found to belong to ST323 [37]. The present study is the second report of an E. faecium ST323 isolate with Q/D resistance associated with bloodstream infection.

Conclusion
In the present study, Q/D-resistant E. faecium clinical isolates were first described in China. We also identified that carriage of vatE, ermB and msrC was responsible for Q/D resistance in E. faecium clinical isolates.