Molecular Characterization of Vancomycin-Resistant Enterococci in a Chinese Hospital Between 2003 and 2009

Abstract

From June 2003 to December 2009, 98 isolates of vancomycin-resistant enterococci (VRE) were cultured from clinical specimens taken from patients admitted to a 1,500-bed tertiary-care teaching hospital in Beijing, China. Isolates were characterized by pulsed-field gel electrophoresis and multilocus sequence typing. We investigated the structure of the vanA gene cluster and the distribution of the virulence markers esp, hyl, gelE, asa1, and cylA by polymerase chain reaction. Our results indicate that multilocus sequence typing revealed five novel sequence types and one new allele. VRE faecium (VREfm) isolates were heterogeneous in their vanA cluster types and in the presence of virulence genes. We also observed inconsistency between genotype and phenotype in VREfm isolates. The outbreak with VREfm in our hospital appears polyclonal, whereas VRE faecalis characterization indicated dissemination of a particular clone. After 2007, VRE faecalis was completely replaced by VREfm, which has since been the predominant species in our hospital. VRE appears to be in an evolutionary flux in our hospital.

Introduction

Vancomycin-resistant enterococci (VRE) were first isolated in the late 1980s in Europe.^18,30 VRE have rapidly expanded and are now reported as the cause of nosocomial infections globally, with reports from the United States,¹² Europe,²⁴ and Eastern Asia.³ VRE has gained importance as a global public health problem because the organism is difficult to treat; in addition, the few remaining therapeutic options are jeopardized by the emergence of resistance to new classes of antibiotics.^20,25

Most of the enterococci causing human infections have been identified as Enterococcus faecalis and Enterococcus faecium. Historically, E. faecalis was responsible for 90% of all enterococcal infections; however, E. faecium infections in the hospital setting have increasingly been reported since the late 1980s. E. faecium attracts increased attention due to its propensity to acquire multiple antibiotic resistance determinants, especially those encoding glycopeptide resistance and its potential to spread in a nosocomial setting. As described in a previous study, E. faecalis is being replaced by multiresistant clonal complex 17 E. faecium.²⁹ Most VRE faecium (VREfm) isolated from hospital-acquired infections worldwide belong to clonal complex-17 (CC-17).^{1,14,16,31,35} Characteristics of strains in this complex include ampicillin resistance, possession of an array of virulence markers, and an association with hospital outbreaks. The virulence genes are located on a large pathogenicity island (PAI).³² The markers for enhanced epidemicity, genes for the virulence and genes for the vancomycin resistance, are localized on mobile elements and may be mobilized or conjugatively transferred when in the appropriate genetic background.^21,34

Throughout China, the prevalence of VRE has been considered low; however, in Beijing, Chaoyang Hospital has reported a prevalence of VRE that is higher than other sites in China.^9,37 Because of the number of patients that are seen at Chaoyang Hospital every year, and since Chaoyang Hospital might represent similar hospitals in other major cities and provinces throughout China, it is very important that an understanding of the molecular mechanisms underlying the rapid dissemination of VRE are exposed. Here we present a detailed molecular analysis of VRE isolated from June 2003 to December 2009.

Materials and Methods

Hospital setting and bacterial culture conditions

Beijing Chaoyang Hospital is a 1,500-bed tertiary-care teaching hospital. Since the first isolation of VRE from the ascites of a patient in the surgery intensive care unit (ICU) on June 21, 2003, a total of 98 VRE cases have been identified (until December 2009). Most of the VRE isolates were recovered from patients in ICUs, particularly in the surgery ICU and the emergency ICU. Overall, 16 different wards reported at least one case. The most common specimen sources were ascites (23 isolates), followed by urine (17 isolates), sputum (16 isolates), blood (11 isolates), wound pus (10 isolates), catheter tips (9 isolates), and bile (6 isolates); 6 isolates were recovered from other sources. Duplicate isolates from the same patient were not included in the study. Bacteria were identified to the genus level using conventional microbiologic methods.

Antimicrobial susceptibility testing

Minimal inhibitory concentrations (MICs) for isolated enterococci were determined by the agar dilution methods, which were performed and interpreted according to the guidelines established by the Clinical and Laboratory Standards Institute.⁴ A panel of 11 antimicrobial agents was tested: ampicillin, levofloxacin, streptomycin, gentamicin, erythromycin, rifampin, tetracycline, linezolid, chloramphenicol, vancomycin, and teicoplanin. E. faecalis ATCC 29212 was used as control strain.

Polymerase chain reaction genotyping, vanA, vanB, and virulence gene detection

Genomic DNA from each of the VRE isolates was extracted by a simple boiling lysis method for molecular analysis. VRE faecalis (VREfs) and VREfm underwent genotype identification by polymerase chain reaction (PCR) amplification using primers targeting species-specific genes.⁵ The vancomycin-resistant genes, including vanA and vanB, were detected using a multiplex PCR as described previously.²²

Virulence genes of VRE encoding aggregation substance (asa1), gelatinase (gelE), cytolysin (cylA), enterococcal surface protein (esp), and hyaluronidase (hyl) were amplified by multiplex PCR as described previously.³³

Structure of Tn1546-like element

DNA sequences of the Tn1546-like elements containing the vanA operon encoding vancomycin resistance were determined using overlapping PCR. Amplification of internal regions of a Tn1546-like element was performed as described previously.²² All PCR fragments were sequenced and products were confirmed as vanA using a BLAST DNA data base search.

Mutilocus sequence typing

Mutilocus sequence typing was performed according to the scheme described previously.¹³ Internal fragments of seven housekeeping genes for VREfm (adk, atpA, ddl, gdh, gyd, pstS, and purK) and VREfs (aroE, gdh, gki, gyd, pstS,, xpt, and yqil) were amplified by PCR, sequenced, and analyzed using standardized methods (www.mlst.net). Alleles for each housekeeping gene were determined and the combination of allelic profiles was used to determine the sequence type (ST) of each isolate.

Pulsed-field gel electrophoresis typing

Pulsed-field gel electrophoresis (PFGE) was conducted as previously described.⁷ Whole-cell genomic DNA of culture lysed cells representing each isolate embedded in 1% agarose plugs (Bio-Rad, Richmond, CA) were digested with the restriction enzyme SmaI (TaKaRa Biotechnology, Dalian, China) and separated by electrophoresis through 1% pulsed-field-certified agarose (Bio-Rad) using a CHEF-Mapper (Bio-Rad). Electrophoretic switch times of 1–20 seconds were used with a 6 V/cm current and a switch angle of 120° under a constant temperature of 14°C. PFGE patterns were interpreted using the criteria proposed by Tenover et al.²⁸

Results

Species and glycopeptide resistance genotype identification

We examined a total of 98 clinical VRE isolates cultured from patients admitted to Beijing Chaoyang Hospital from June 2003 to December 2009. Of the 98 VRE, there were 81 E. faecium and 17 E. faecalis. Amplification of vanA and vanB showed that the vanA gene was exclusive to E. faecium isolates, whereas vanB gene was unique to E. faecalis isolates.

Antimicrobial susceptibility of VRE isolates

In this study all VRE isolates were resistant to erythromycin, levofloxacin, and vancomycin. However, only the 81 VREfm isolates were resistant to ampicillin; isolates of VREfs were susceptible to this antibiotic. Only 9.3% of the isolates were resistant to tetracycline and 6.2% were resistant to chloramphenicol. All isolates were sensitive to linezolid. The vancomycin MIC for VREfm isolates was high (>256 μg/ml); however, 17 (21%) of these isolates were susceptible to teicoplanin. This result demonstrates an incongruence of the VanB phenotype with the vanA genotype in these 17 VREfm isolates.

Structure of Tn1546-like element

Analysis of the central region of the Tn1546 elements after PCR and DNA sequencing showed no size variation or sequence polymorphisms. The vanRSHAX genes were conserved in all 81 VREfm isolates. However, the remaining parts of Tn1546, including the right and left ends, were heterogeneous and had the integration of insertion sequence (IS), accompanied by deletions adjacent to the insertion sites. Based on the Tn1546 element sequence, the 81 VREfm isolates were classified into four different transposon types, including eight subtypes of vanA clusters related to the prototype Tn1546. Transposon type A was the most predominant (n = 28), followed by type D (n = 27), type C (n = 16), and type B (n = 10). Transposon type A had a copy IS1542 insertion in the orf2-vanR intergenic region and a copy insertion of IS1216V in vanX-vanY intergenic region, accompanying with partial deletion of the downstream insertion site of IS1216V, but both vanY and vanZ gene were complete. Transposon type B had similar insertion as transposon type A, but it accompanied complete deletion of vanY and vanZ. Transposon type C had a copy IS1542 insertion in the orf2-vanR intergenic region. Transposon type D had similar insertion as transposon type A, but it had an ISEfa4 element 66 bp downstream of IS1542 in the vanA gene cluster. On the other hand, all of isolates with a type B Tn1546 structure phenotypically expressed the VanB phenotype but were a vanA genotype. Four additional isolates expressed the VanB phenotype-vanA genotype and harbored type D transposons. The remaining three VanB phenotype-vanA genotype VREfm isolates harbored type A transposons.

Multilocus sequence typing and PFGE genotyping

We used a combination of PFGE and multilocus sequence typing (MLST) to provide the molecular characteristics of the 17 VREfs (Fig. 1) and 81 VREfm (Fig. 2) isolates. MLST analysis identified 18 STs, including 5 newly identified STs (ST555, ST569, ST570, ST571, and ST572) and a novel allele (AtpA59). The VREfs isolates were indistinguishable; all 17 isolates were genotyped as ST4. In contrast, 17 STs were present among the VREfm isolates. The most prevalent ST in VREfm was ST17 (n = 28; 34.6%), followed by ST78 (n = 15; 18.5%), ST290 (n = 6; 7.4%), ST414 (n = 5; 6.17%), ST572 (n = 5; 6.17%), ST203 (n = 4; 4.94%), ST363 (n = 4; 4.94%), ST192 (n = 3; 3.7%), ST343 (n = 2; 2.5%), and ST18 (n = 2; 2.5%). Seven STs were represented by only a single isolate.

FIG. 1.

Characteristics of investigated Enterococcus faecalis isolates. Pulsed-field gel electrophoresis of SmaI-digested genomic DNA.

FIG. 2.

Characteristics of investigated Enterococcus faecium isolates. Pulsed-field gel electrophoresis of SmaI-digested genomic DNA.

Our results indicate that the 98 isolates belonged to a minimum of 33 different PFGE profiles (the banding patterns are designated PFGE 1 to 33; Figs. 1 and 2). The predominant profiles were 1, 33, and 23, accounting for 26.5% (26/98), 17.4% (17/98), and 8.2% (8/98) of strains, respectively. Similar to the results obtained by MLST, the 17 VREfs isolates appeared clonal; there was a single dominant profile with single- and double-band variants present within the PFGE profiles (PFGE 33). None of the VREfm isolates had this PFGE profile. The remaining PFGE profiles were present within only VREfm isolates, once again demonstrating the genetic diversity among these isolates. We did observe, however, inconsistencies between the two genetic methods. VREfm isolates falling in the same ST showed different PFGE patterns. For example, of the 28 ST 17 isolates, we observed 4 PFGE types (1, 6, 10, 15). We also found VREfm isolates that had indistinguishable PFGE profiles (e.g., 8, 1) that had unique STs (ST78 and ST17, respectively).

Determination of virulence markers

All 17 VREfs harbored the virulence genes esp, gelE, asa1, and cylA. However, the hyl gene was not detected (Fig. 1). Consistent with the heterogenous nature of the VREfm observed with MLST and PFGE, we were able to amplify esp from 72 isolates (88.9%), hyl from 35 isolates (43.2%), and gelE from 33 isolates (40.7%), respectively (Fig. 2). We were unable to amplify the cylA and asa1 genes from any VREfm isolate.

Discussion

In the present study, VREfm isolates were heterogeneous in their vanA cluster types and in the presence of virulence genes. MLST revealed five novel STs types and one new allele. We also observed incongruence between genotype and phenotype in VREfm isolates. The outbreak with VREfm in our hospital appears polyclonal, whereas VREfs characterization indicated dissemination of a particular clone. After 2007, VREfs was completely replaced by VREfm which has since been the predominant species in our hospital.

It is clear that vanA resistance elements are heterogeneous. The heterogeneity of the Tn1546-like element is mostly associated with the IS elements. For example, the insertion of IS1251 in the vanS-vanH intergenic region was described in VREfm from the United States, Brazil, Poland, and Russia.^1,2,10,15 The insertion of IS1542 in the orf2-vanR intergenic region and IS1216V in the intergenic region of vanX-vanY has previously been detected in VREfm isolates from Korea.¹⁹ In the present study the majority of our isolates were characterized by an IS1542 insertion in the orf2-vanR region and an IS1216V insertion in vanX-vanY intergenic region. This is similar to the situation described in Korea. An additional 27 (33.3%) VREfm isolates from our hospital contained an ISEfa4 66 bp downstream of the IS1542, located in the orf2-vanR intergenic region of the vanA gene cluster. ISEfa4 was initially found in vanS gene of vanD genotype VRE.⁶ However, ISEfa4 was found in the orf2-vanR intergenic region of the vanA gene cluster from our collection.⁹ Our result suggested that ISEfa4 at the left end of Tn1546 was acquired later than IS1542 and indicate the ongoing evolution of the E. faecium population.

As described in a previous study, the esp gene is a marker of a highly prevalent nosocomial E. faecium lineage.³⁶ VREfm isolates from our collection showed a heterogeneous virulence gene pattern (Fig. 2). Different STs (ST-343, ST-203, ST-363, ST-389, ST-192, ST-252, ST-555, and ST-570) possess an identical virulence profile in which all were esp-positive and hyl-negative. However, in other STs (ST-17, ST-290, ST-78, ST-414, ST-18, and ST-572) virulence genes appeared to be randomly distributed. For example, of the 28 ST-17 isolates, 13 possessed only the esp gene, 12 were positive for esp and hyl, 1 harbored only hyl, and 2 lacked both genes. The prevalence of the esp gene within our 81 VREfm isolates was 88.9%. Although reports of esp prevalence vary according to region and population, our results are consistent with those of Brilliantova et al., who found the prevalence of esp to be 91% in E. faecium isolates studied in Russia.¹ The hyl gene is also associated with clinical E. faecium isolates, with a ratio of 2:1 for esp: hyl-positive isolates reported in a sample of 577 isolates worldwide.²³ This compares closely with the ratio of 2.1:1 obtained from our smaller collection. The prevalence of the gelE gene (40.7%) is somewhat lower than previously reported.¹ We were unable to amplify the cylA and asa1 genes from any VREfm isolate. This is consistent with other studies that have not shown detection cylA and asaI in E. faecium.⁸ In a recent study, it was found that esp is carried within a large PAI and the esp PAI is mobilizable.³² Gain and loss of mobile genetic elements is the most important driving force in determining virulence-associated properties in epidemic strains.³² Acquisition of potential virulence traits by epidemic strains might enhance their ability to spread and cause infection and increase their fitness in the hospital environment.

Reports from several Asian countries, mainly, Japan, South Korea, and China, have documented VREfm isolates demonstrating the VanB phenotype-vanA genotype. These reports have indicated that point mutations and/or rearrangements to the vanS, vanX, vanY, and van Z genes are responsible for this incongruence.^{9,11,17,19,26} In the present study, 64 (79%) of the 81 VREfm with the vanA genotype showed the VanA phenotype, whereas 17 (21%) of the 81 VREfm with vanA genotype showed the VanB phenotype. The observed prevalence of VREfm isolates with the VanB phenotype-vanA genotype was higher in comparison than reported by a previous Korean study.²⁷ Based on the PCR mapping of Tn1546 elements, three transposon types were associated with the VanB phenotype-vanA genotype in this study. All type B transposons (10 isolates) showing the deleted form of vanY and vanZ genes belong to the VanB phenotype-vanA genotype, supporting previous observations that deletion of the vanY and vanZ genes is directly associated with this incongruence. Four additional isolates expressed the VanB phenotype-vanA genotype and harbored type D transposons. We could not find distinct incongruence mechanisms through current experiment. The remaining three VanB phenotype-vanA genotype VREfm isolates harbored type A transposons. Our analysis also failed to reveal a common change that could explain the VanB phenotype. These results suggested that the occurrence of VanB phenotype-vanA genotype VREfm isolates might not be due to a single genetic variation. Further investigation is required to elucidate the molecular mechanism of the VanB phenotype-vanA genotype in these isolates; the clinical importance of these strains is under investigation.

MLST analysis revealed that the predominant strain was ST17, which is the predicted founder of CC17. We also identified several new STs. Among the five novel ST types, four alleles of single locus variants have arisen by recombination. This result suggested that the recombination played an important role in genetic diversification in E. faecium in our hospital. All isolates characterized as ST572, a previously unreported ST type, had the same transposon B pattern with incongruence between phenotype and genotype. This strain has been increasingly isolated in the recent years in our hospital.

All VREfs isolated in the present study possess the vanB genotype; VREfs-containing vanB were from different wards in the Chaoyang Hospital and exhibited a single dominant profile with single and double band variants present within the PFGE profiles (PFGE 33), and a uniform MLST type (ST4), indicating dissemination of a particular clone. We have not isolated this clone since February 2007. However, all the VREfm in the present study possess a vanA genotype. PFGE results revealed a polyclonal distribution of vanA VREfm isolates, indicating that strains have originated from several different sources. The predominant PFGE type was 1. The prevalence of vanA-containing VREfm strains is increasing in our hospital. Since 2007 VREfs was completely replaced by VREfm.

Considering the heterogeneous of the Tn1546-like element and the virulence gene patterns within identical ST, the appearance of new ST and transposon types, we conclude that the E. faecium population is under constant selection and continues to evolve in response to changes in the hospital environment.

Footnotes

Acknowledgments

The authors thank Huaxia Chen, Shoushan Qu, Fang Li, binbin Li, ShanshanWang, Ping Guo, Chunxia Yang, and Chunlei Wang for their assistance. We also thank Dr. John Klena for his comments. This work was financially supported by National Natural Science Foundation of China (30870094/C010603) for Dr. Bin Cao.

Disclosure Statement

No competing financial interests exist.

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