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IS element IS16 as a molecular screening tool to identify hospital-associated strains of Enterococcus faecium

  • Guido Werner1Email author,
  • Carola Fleige1,
  • Uta Geringer1,
  • Willem van Schaik2,
  • Ingo Klare1 and
  • Wolfgang Witte1
BMC Infectious Diseases201111:80

DOI: 10.1186/1471-2334-11-80

Received: 6 May 2010

Accepted: 31 March 2011

Published: 31 March 2011

Abstract

Background

Hospital strains of Enterococcus faecium could be characterized and typed by various molecular methods (MLST, AFLP, MLVA) and allocated to a distinct clonal complex known as MLST CC17. However, these techniques are laborious, time-consuming and cost-intensive. Our aim was to identify hospital E. faecium strains and differentiate them from colonizing and animal variants by a simple, inexpensive and reliable PCR-based screening assay. We describe here performance and predictive value of a single PCR detecting the insertion element, IS16, to identify hospital E. faecium isolates within a collection of 260 strains of hospital, animal and human commensal origins.

Methods

Specific primers were selected amplifying a 547-bp fragment of IS16. Presence of IS16 was determined by PCR screenings among the 260 E. faecium isolates. Distribution of IS16 was compared with a prevalence of commonly used markers for hospital strains, esp and hyl Efm . All isolates were typed by MLST and partly by PFGE. Location of IS16 was analysed by Southern hybridization of plasmid and chromosomal DNA.

Results

IS16 was exclusively distributed only among 155 invasive strains belonging to the clonal complex of hospital-associated strains ("CC17"; 28 MLST types) and various vancomycin resistance genotypes (vanA/B/negative). The five invasive IS16-negative strains did not belong to the clonal complex of hospital-associated strains (CC17). IS16 was absent in all but three isolates from 100 livestock, food-associated and human commensal strains ("non-CC17"; 64 MLST types). The three IS16-positive human commensal isolates revealed MLST types belonging to the clonal complex of hospital-associated strains (CC17). The values predicting a hospital-associated strain ("CC17") deduced from presence and absence of IS16 was 100% and thus superior to screening for the presence of esp (66%) and/or hyl Efm (46%). Southern hybridizations revealed chromosomal as well as plasmid localization of IS16.

Conclusions

This simple screening assay for insertion element IS16 is capable of differentiating hospital-associated from human commensal, livestock- and food-associated E. faecium strains and thus allows predicting the epidemic strengths or supposed pathogenic potential of a given E. faecium isolate identified within the nosocomial setting.

Background

Vancomycin- and multi-resistant Enterococcus strains, especially strains of E. faecium, raise major concerns in intensive care medicine due to limited treatment options [1]. E. faecium is ecologically widely distributed and expected to play a central role as a reservoir and "turn-table" for antibiotic resistance determinants in the bacterial world, and especially among Gram-positive bacteria [2]. A number of molecular typing and characterization techniques such as AFLP, MLVA and MLST and comparative genomic hybridizations allowed differentiating strains of E. faecium and allocating them into various clonal complexes as based on core as well as accessory genome content [3, 4]. Isolates belonging to hospital-associated clonal types (for instance, MLST CC17) could be identified and a supposed enhanced spreading potential among the nosocomial setting for isolates of this specific subgroup is predictable [4]. Reports from recent years described increasing annual rates of E. faecium bacteraemia in European countries [5, 6]. When investigated in greater detail, the increase was due to increasing numbers of hospital-associated clonal types of E. faecium (MLST CC17) whereas numbers of other clonal types remained constant over time again emphasizing the aforementioned increased potential for nosocomial spread [5]. The molecular techniques to identify hospital-associated E. faecium strains are laborious, time-consuming and cost-intensive and are thus not applicable for routine diagnostics. Results of recent bacterial, epidemiological, microarray-based and genomic studies suggest that a number of markers are enriched among hospital strains of E. faecium including collagen adhesion factors acm and scm [7, 8], other matrix-binding proteins and pili [9, 10], a supposed hyaluronidase, hyl Efm [11, 12], biofilm-associated markers including the enterococcal surface protein gene, esp [13, 14], and various genomic islands encoding proteins of unknown functions [15, 16]. Having acquired a number of these aforementioned determinants, these strains may have an increased pathogenic potential [4, 10, 17]. However, distribution of these markers among hospital strains is not exclusive and thus the predictive value is limited in terms of specificity and sensitivity. Certain markers possess pseudogenes and partly deleted variants complicating establishing a simple PCR based screening test [8, 17]. Acquired ampicillin resistance appeared as a phenotypic trait of hospital strains, at least in Europe; however, this feature is also prevalent among non-hospital strains in some parts of the world, such as Asia and North America [1820]. Microarray based genomic comparisons revealed a specific mobile element, insertion element IS16, exclusively prevalent among hospital E. faecium strains from an international collection of isolates [3]. In the present study, we established a PCR-based IS16 screening test and evaluated its robustness, sensitivity, specificity and predictive value for 260 pre-characterized isolates of E. faecium from different human and animal sources. IS16 was localized in a subset of E. faecium isolates (n = 45) representing 29 MLST types by Southern hybridization. Available E. faecium genome data were examined for the presence of IS16 homologous sequences and a genetic association of IS16 with genomic markers is discussed.

Methods

Strain collection

Our laboratory serves as a Focal Laboratory for Enterococci in Germany. In general, enterococcal isolates sent to us are pre-selected for being vancomycin- and multi-resistant or with the suspicion of clonal spread (local, regional, and country-wide outbreak analysis). Our collection of E. faecium test strains included 160 invasive isolates (blood cultures) from 60 different German diagnostic or hospital laboratories isolated between 1995 and 2007. Blood culture strains were pre-selected by geography and hospital origin, year of isolation, and different van genotypes (van-/A/B). The blood culture isolates were compared against 68 human commensal (outpatient) isolates from four different prevalence studies performed in different parts of Germany (1996, 1997, 1999, 2003) [24, 25] and 32 isolates from animal (chicken, pigs) and meat samples (broiler, pork) (1994, 1995, 1999) [2123]. In all cases duplicate isolates were excluded based on clinical/epidemiological data (same patient/hospital or animal sample/flock) and of molecular typing results (for instance, based on SmaI-macrorestriction using PFGE for human commensal isolates [24, 25]; unpublished results). Antibiotic susceptibilities were determined by microbouillon dilution in MH bouillon. Ampicillin resistance is defined by an MIC of >8 mg/L and vancomycin resistance by >4 mg/L (http://www.eucast.org). Summarized data are presented in Table 1 and detailed information is given in Additional files 1, 2, 3. Ethical approval was not needed for this study; all strains were pre-selected and were received anonymously from German microbiological or hospital laboratories.
Table 1

Prevalence of epidemic markers among E. faecium isolates from different origins (see also Figure 1 and Additional Files 1, 2, 3).

Origin

No. of isolates

No. of MLST types

AmpR

PCR IS16+

PCR esp+

PCR hyl Efm +

PCR vanA+/B+

Blood cultures

160

32

98.1% (n = 157)

96.9% (n = 155)

63.8% (n = 102)

45.6% (n = 73)

58.1% (n = 93)

   Hosp.-associated E. faecium (CC17) 1

155

28

100% (n = 156)

100% (n = 156)

65.8% (n = 102)

46.5% (n = 72)

58.7% (n = 91)

Human commensal

68

46

2.9% (n = 2)

4.4% (n = 3)2

0%

0%

14.7% (n = 10)

Animal commensal and meat sources

32

22

6.2% (n = 2)

0%

0%

0%

40.6% (n = 13)

BC MLST CC17 (second line) shown in italics (n = 155) is a subgroup of all blood culture E. faecium isolates presented in the first line representing all isolates allocated to the clonal complex of hospital-aascoiated strains (CC17).

AmpR, ampicillin resistance; 1 including two isolates of ST65 representing a singleton but known to be found only among clinical strains; 2 all three belonged to the clonal complex of hospital-associated strain types (MLST CC17; 2x ST18, 1x ST413; see also Additional file 4).

DNA isolation

Genomic DNA was prepared using a DNA extraction kit (DNeasy Tissue Kit; Qiagen, Hilden, Germany) according to the manufacturer's instructions. An initial cell wall lysis step was added dissolving the cell pellet in TES buffer [10 mM Tris, 0.5 mM ethylene diamine tetra-acetic acid (EDTA), 10% sucrose (pH 8.0)] plus 10 mg/mL lysozyme (Roche Applied Science, Mannheim, Germany) followed by incubation at 37°C for 30 min. Plasmid DNA was isolated using a common phenol/chloroform-based alkaline lysis strategy [26].

PCR

PCR was performed with illustra™ PuRe Taq Ready-To-Go™ PCR Beads (GE Healthcare, Freiburg, Germany) according to the manufacturer's instructions. Approximately 0.5 μL of isolated genomic DNA (ca. 10 ng) and primers (200 nM each) were added. Amplification of fragments representing the esp, hyl Efm and vanA/B genes was performed in a multiplex PCR as described elsewhere [27]. The following strains and plasmids were used as positive control samples: plasmid pRUM (IS16), plasmid pLG1 (hyl Efm ); plasmid pIP816 (vanA; E. faecium BM4147), E. faecium U0317 (esp), and E. faecalis V583 (vanB). E. faecalis OG1RF served as a negative control sample for all PCR assays. PCR primers and conditions for screening IS16 elements were as follows: IS16-F: 5'-CATGTTCCACGAACCAGAG and IS16-R: 5'-TCAAAAAGTGGGCTTGGC, annealing temperature was 53°C, the predicted size of the product was 547 bp (reference E. faecium plasmid pRUM; GenBank acc. no. AF507977). Primers were established using software Acelrys DS Gene 1.5.

PFGE typing

Genomic DNA for PFGE analysis was isolated and treated as described recently [27]. The agarose gel concentration was 1%, the CHEF-DR III apparatus (Bio-Rad Laboratories, Hercules, CA, USA) was used for PFGE. The ramped pulsed times were as follows: 1 - 11 sec for 15 h and 11 - 30 sec for 14 h at 14°C. SmaI-digested Staphylococcus aureus NCTC 8325 was used as a molecular mass standard on all PFGE gels. Chromosomal DNA of E. faecium strains was digested with I-Ceu-I for 16 h at 37°C. Plasmids were linearized by S1 nuclease (TaKaRa, Germany) treatment for 15 min (37°C) and subsequent resolution of the linearized plasmid DNA in PFGE. The ramped pulsed times for I-Ceu-I gels were 5 - 30 sec for 22 h and for S1 PFGE 5 - 35 sec for 22 h at 14°C [28].

Southern hybridizations

Southern hybridization experiments were done as described elsewhere using a PCR-generated digoxigenin-labelled IS16 probe (DIG High Prime; Roche Applied Science, D), hybridization chemicals and equipment from commercial kits and according to recommendations of the manufacturer (Roche, Germany). Immunological detection was done as recommended using a chemiluminescent probe (CDP-Star™, Roche, D) and several readouts were taken at 10, 30, 60 and 120 min in a chemi-imager from Bio-Rad (Chemidoc XRS, Bio-Rad Labs., Hercules, US).

MLST and DNA sequencing

PCRs amplifying the seven loci used for MLST were done according to the reference (http://efaecium.mlst.net/). Sequencing reactions were performed according to the manufacturer's recommendations for cycle sequencing of PCR products (Applied Biosystems, Germany). Sequence files were read, evaluated, aligned and compared to the reference set of alleles using sequencing software Lasergene 8.0 from DNA-STAR (SeqMan 8.0; EditSeq 8.0), TraceEditPro v. 1.1.1 from Ridom (http://www.ridom.de), and via the official MLST webpage (http://efaecium.mlst.net/). Phylogenetic trees were generated using software programmes eBURST [29] for the set of determined MLST types and global optimised eBURST [30] for the entire set of all available 524 MLST types (http://efaecium.mlst.net/).

Statistics

Statistical analyses were performed with software package EpiCompare 1.0 (Ridom).

Results

Identification of IS16primer sequences

Based on available sequences (pRUM [31] or composite (vanB) transposons [32]) we established PCR primers for an IS16-specific internal fragment (547 bp). Specificity and sensitivity was evaluated against available bacterial databases (BlastN, GenBank). The PCR was established using DNA from reference samples known to be positive for IS16 (plasmid pRUM) and from negative control samples known to lack IS16 (E. faecalis OG1RF). PCR results were as expected and amplified products from reference samples were sequenced for confirming specificity of the amplified PCR product (not shown).

Screening for markers in the two sets of strains

Genomic DNA was screened by PCR using the established PCR primers for IS16, esp and hyl Efm . IS16 was highly prevalent among invasive E. faecium (n = 155/160; Table 1) and mainly absent (n = 3/100) from human commensal or animal and meat sources. The three IS16-positive commensal isolates were from humans. Other markers supposedly enriched among clinical or hospital-associated E. faecium isolates were also assessed: the esp gene was prevalent among 64% of all blood culture isolates and 46% of all invasive isolates were hyl Efm -positive (Table 1). None of the 100 non-clinical strains possessed esp or hyl Efm . Ampicillin resistance was prevalent among almost all clinical strains (98%). Two human commensal and two animal isolates were also ampicillin-resistant.

MLST typing

All 260 E. faecium isolates were MLST typed. Discriminatory power among clinical isolates (n = 160 isolates; 32 MLST types DI = 0.889 95% CI [0.864 - 0.913]) was much less than among human and animal commensal isolates (n = 100 isolates, 64 MLST types; DI = 0.988; 95% CI [0.983 - 0.994]). This suggests that prevalent hospital-associated clonal types differ less than their colonizing counterparts in animals and humans. In fact, 126 of the 160 invasive E. faecium isolates belonged to only eight MLST types known to represent major hospital-associated clonal types (ST16, ST17, ST18, ST78, ST117, ST192, ST202, ST203) whereas the other 24 MLST types occurred only occasionally. Based on their MLST types and subsequent eBURST and goeBURST analyses, isolates could be sorted into different clonal complexes which were in agreement with their corresponding ecological background (compare also to [4]). Figure 1 shows eBURST trees generated with all MLST types determined in this study. A phylogenetic tree of all currently detached MLST types (n = 524; 21.04.2010; http://efaecium.mlst.net/) using goeBURST is shown as Additional File 4. Based on these two calculations, almost all (n = 155/160) invasive isolates belonged to the clonal complex of hospital-associated strains (CC17 + ST65 [singleton]). The five IS16-negative clinical isolates represent colonizing strain types not associated with clonal types of hospital strains ("non-CC17"; ST9 [n = 2], ST104; ST271; ST374). The majority of commensal isolates represented many individual MLST types but belonging to the major clonal complexes of animal and human colonizing strain types (CC5, CC22/36, CC1). The three IS16-positive commensal E. faecium isolates revealed MLST types (ST18, ST413) belonging to the clonal complex of hospital-associated strains (CC17).
https://static-content.springer.com/image/art%3A10.1186%2F1471-2334-11-80/MediaObjects/12879_2010_Article_1418_Fig1_HTML.jpg
Figure 1

eBURST cluster of MLST types generated from this set of 260 clinical, commensal and livestock-associated E. faecium strains. (The newly identified MLST types are not presented as well as the following MLST types which appear as singletons in this set: ST39, ST74, ST94, ST289, ST345, ST503, ST413; ST512; compare also to Additional file 4). The origin of the isolates is given in colour codes. ST19 appears here as a singleton but belongs to CC17 when resolved among the entire set of MLST types (Additioinal file 4).

Southern hybridizations of IS16probes

The localization of IS16 among 45 representative E. faecium isolates from various origins and altogether 29 different MLST types was investigated. I-Ceu-I digested chromosomal DNA resolved in PFGE and plasmid DNA analysed inS1 PFGE and common agarose gels were transferred onto nylon membranes and hybridized against a labelled IS16 probe. Results are shown in Table 2. If available, several representatives of identical MLST types differing in their van gene content were considered for analysis (see for instance ST17 vanA/B/-negative isolates). In general, the IS16 probe hybridized to (up to three) chromosomal bands suggesting several copies of the same element present in the genome (a single hybridizing chromosomal band could still result from several IS16 copies on it). Four isolates (ST17, ST81, ST202, ST275) showed IS16 only hybridizing to plasmid bands. Twenty-two strains (n = 22/45) revealed a signal for both chromosomal as well as plasmid bands. Hybridization patterns were independent of the van gene content (Table 2).
Table 2

Localization of IS16 to I-Ceu-I digested chromosomal DNA resolved in PFGE and plasmid DNA as based on Southern hybridizations.

Strain no.

Origin

MLST type

vangenotype

I-CeuI PFGE

Plasmid

UW 6714

BC

202

-

-*

+

UW 1245

BC

202

vanA

-

+

UW 6293

BC

202

vanB

-*

-#

UW 6295

BC

202

vanB

-*

-#

UW 4962

BC

203

vanA

+

-#

UW 6989

BC

203

-

+

-#

UW 6502

BC

208

vanB

+

+

UW 5918

BC

252

vanA

+

+

UW 4675

BC

252

vanB

+

-

UW 6617

BC

275

vanA

-

+

UW 4960

BC

279

vanB

+

-

UW 6033

BC

282

vanA

+

+

UW 4512

BC

294

-

+

+

UW 6297

BC

306

-

+

+

UW 6935

BC

371

-

+

+

UW 3142

BC

375

vanA

+

+

UW 5275

BC

376

vanB

+

+

UW 6983

BC

377

-

+

+

UW 6112

BC

378

-

+

+

UW 4208

BC

New

-

+

+

UW 6642

BC

New

vanA

+

+

AK-EM 40

HC

18

-

+

+

AK-EM 53

HC

413

-

+

+

K226.7

HC

18

-

+

-

UW 2055

BC

16

vanA

-*

+

UW 3695

BC

17

-

+

+

UW 3544

BC

17

vanA

+

-#

UW 5352

BC

17

vanB

-

+

UW 6990

BC

18

-

+

+

UW 1952

BC

18

vanA

+

+

UW 2900

BC

18

vanB

+

-#

UW 1218

BC

19

vanA

+

-#

UW 3056

BC

65

-

+

+

UW 6880

BC

78

-

+

+

UW 4671

BC

78

vanA

+

-

UW 2457

BC

80

vanA

+

-

UW 6985

BC

81

-

-

+

UW 2771

BC

81

vanA

+

-

UW 6882

BC

117

-

+

-

UW 1983

BC

117

vanA

+

-

UW 6805

BC

125

vanA

+

+

UW 2824

BC

132

vanA

+

-#

UW 1716

BC

192

-

+

-#

UW 5852

BC

192

vanA

+

+

UW 5267

BC

192

vanB

+

-#

If available, different representatives of identical MLST types were considered (see for instance ST17 vanA/B/-negative isolates).

PFGE, pulsed-field gel electrophoresis; MLST, Multi-locus sequence typing; BC, blood culture; HC, human commensal; *, Hybridization signal with PFGE slot; #, negative results in repeated experiments.

Screening E. faecium genomes for IS16

Several E. faecium genomes are available as genomic fragments for preliminary analysis (collection I: seven E. faecium genomes; [33]; collection II: eleven E. faecium isolates from the Human Microbiome Project; (http://nihroadmap.nih.gov/hmp/; http://www.ncbi.nlm.nih.gov//genomes/geblast.cgi?gi=6511#SearchSet). A BLAST search for IS16-related sequences in both E. faecium collections revealed several hits. However, as far as some detailed information was available, the neighbouring DNA fragments all showed different genetic links. IS16 was prevalent among four out of seven genomes in collection I [33]. IS16 was linked to (a) several putative fragments of a conjugative transposon (strain E1162, ST17), (b) ORF involved in sugar metabolism (strain E1636; ST106), (c) a genomic fragment containing other transposases of the IS256 family (strain U0317, ST78) and (d) the supposed pathogenicity island containing the esp gene (strain E1679, ST104). None of the other two strains also containing the esp PAI possessed IS16 within its structure. In the deposited genomic fragments from the Human Microbiome Project, IS16 was infrequently identified, for instance, in E. faecium 1,230,933 linked to a metal-dependent hydrolase or in E. faecium TC6 linked to an extracellular solute-binding protein, family 1. In E. faecium DO a 5' link to a C4-dicarboxylate anaerobic carrier gene and a 3' link to a chloramphenicol actetyltransferase resistance gene followed by a replicase and relaxase genes indicated a supposed plasmid origin of this contig (not shown in details). Available sequence data allow extracting the MLST type related allele information out of the sequenced genomes and a corresponding analysis was done on the seven (collection I) and eleven (collection II) accessible genomes. Of the 11 isolates positive for IS16 eight strains possessed hospital-associated MLST types (ST16, ST17, ST18, ST78, ST203, ST-NEW) and three did not belong to clonal complexes of hospital-associated types (ST25, ST104, ST114). Intriguingly, of the latter three, one was a clinical isolate also positive for esp, a marker commonly associated only with hospital-associated strains.

Epidemiological analysis of clinical strains

We introduced PCR-based screening for IS16 for all E. faecium isolates sent to our Focal Laboratory for Enterocooci in Germany in late 2007. In 2008 we received 314 E. faecium isolates (from >57 hospitals located in 13 of the 16 German federal states; all VRE) and in 2009 334 E. faecium isolates (from >49 hospitals located in 11 German federal states, 90% VRE) from clinical sources for further analyses. Altogether 99.4% (n = 312/314) in 2008 and 97.3% (n = 325/334) in 2009 were IS16-positive demonstrating wide prevalence of multi-resistant (VRE), hospital-associated strain types in hospitals in Germany.

Discussion

Early recognition of epidemic E. faecium strains is critical for a timely introduction of strategies to prevent and control their further spread. Hospital-associated clonal types of E. faecium often disseminate unrecognized among the nosocomial setting before and until first VRE cases suddenly appear [5, 6]. But standardised methods for rapid diagnostics of these hospital-associated E. faecium strains are missing and modern, DNA sequence-based typing techniques are laborious, time-consuming and comparably expensive. Acquired ampicillin resistance [5, 6, 34] and high-level ciprofloxacin resistance [35, 36] appear as phenotypic traits of hospital-associated strains. However, ampicillin resistance in animal or commensal enterococci is also prevalent to a certain extent [37], at least in many North American and Asian countries [38] and the predictive value of acquired ciprofloxacin resistance is limited since it accumulates over time and differently in various clonal types of hospital strains [36]. Several genetic markers, such as esp, hyl Efm or the purK1 allele (used as part of the MLST scheme) are not ubiquitous traits of all hospital-associated E. faecium strains and failure to detect them does not reliably indicate a strain with a limited spreading or pathogenic potential [27, 34, 36, 3941]. It was also shown recently that hyl Efm is plasmid-located [11, 42], transferable in vitro into several non-hospital E. faecium strains and thus cannot be suggested to serve as a specific marker to predict a clonal background of a given strain [43]. A reliable and rapid molecular test to differentiate commensal from hospital-acquired strains is desirable; results from comparative genomic hybridizations and genome sequencing projects show promising candidate markers including the described insertion element IS16 [3].

According to our data, IS16 is highly specific for predicting hospital-associated strain types (Table 1; Additional files 1, 2, 3). Altogether 155 of the 160 blood culture isolates from 1995 to 2007 from >50 contributing laboratories or hospitals and representing 28 MLST types were all IS16 positive. The comparably high number of (a) participating hospitals/laboratories contributing strains, (b) blood culture strains representing many different MLST types and different van genotypes, and (c) isolated from over 13 years compensates for a geographical limitation of a strain collection that only included isolates from Germany. The presence of IS16 was independent of resistance gene clusters known to possess this element; only 19 isolates were vanB-positive (12%) and the remaining isolates were vancomycin susceptible (n = 67; 42%) or of the vanA-type (n = 74; 46%). Since it is known from previous plasmid analysis that IS16 could be associated with specific plasmid types (e.g., pRUM), we identified the location of IS16 in a representative set of E. faecium isolates (vanA/B/-negative; 29 MLST types; Table 2). Southern hybridizations revealed chromosomal, plasmid and both localizations for IS16 suggesting a somehow restricted mobile gene pool within hospital-associated clonal types. All but three investigated human commensal and all animal strains possessed MLST types different from hospital-associated strains; all but the three aforementioned strains were IS16 negative. The three human commensal isolates possessing IS16 revealed MLST types ST18 and ST413, allocating them to hospital-associated strains and again emphasising the excellent predictive value of this element for identifiying hospital-associated strain types. PCRs for esp and hyl Efm were negative for these three strains and one was ampicillin-resistant. Absence of IS16 among non-clinical E. faecium strains also suggests a very limited host range of IS16-bearing plasmids (e.g., pRUM-like) only among hospital-associated strains. Prevalence of hospital-associated E. faecium isolates among the general human population is low and more detailed information was only available in one of the three cases. This outpatient from a community surveillance study received beta-lactam treatment during the time of sampling which could be the reason for identifying an ampicillin- and multiresistant, epidemic strain here (association with a hospital stay was not documented).

An epidemiological analysis of 648 E. faecium clinical isolates sent to our Focal Laboratory for Enterococci in 2008/2009 from German hospital patients revealed that 98% (n = 637) were IS16-positive, demonstrating country-wide prevalence of hospital-associated strain types among German hospitals in recent years. This finding was supported by wide prevalence of acquired high-level ciprofloxacin resistance among the same strain collection serving as an additional marker for hospital-associated strains [36]. Admittedly, most of the strains from 2008/2009 were vancomycin-resistant showing in retrospect that hospital-associated strains of E. faecium and not commensal strain types accumulated in the nosocomial setting and acquired vanA- or vanB-type resistance determinants.

The observation that an IS element, IS16, is among the most specific markers to define hospital-associated strains [3] appears curious regarding the promiscuous nature of these elements to transpose between several genomic backgrounds within a cell and the possible horizontal mobilization in cis or trans by activation via conjugative elements and subsequent transfer into new hosts. It has been argued that acquisition of specific elements, especially IS16, has contributed to the ecological success of hospital-associated E. faecium strains allowing higher levels of genetic variability including easier acquisition of mobile genetic elements that may support lifestyle in new or changing environments or ecological niches like the hospital environment [3, 44].

Conclusions

Our data show that an IS16-based screening assay appears as the best molecular screening tool to differentiate colonizing E. faecium isolates from hospital-associated E. faecium strains that show an enhanced spreading potential in the nosocomial setting. The predictive value of an IS16 result is superior to the predictive strengths of established tests targeting molecular markers like esp or hyl Efm or phenotypic traits like acquired ampicillin or high-level ciprofloxacin resistance. Progress in comparative genome-based analysis will most probably reveal additional markers to identify hospital associated E. faecium isolates with an enhanced spreading potential in the nosocomial setting [15, 33].

Declarations

Acknowledgements

We kindly acknowledge all colleagues working in medical-microbiological laboratories and hospital laboratories sending their samples for further analyses to the Focal Laboratory for Enterococci, Germany, and apologize for being unable to list them all here. The study was partly supported by grants from EC-funded projects ("ACE", LSHE-CT-2007-037410; "TROCAR", LSHE-CT-2008-223031).

Authors’ Affiliations

(1)
Robert Koch Institute, Wernigerode Branch
(2)
University Medical Centre

References

  1. Arias CA, Murray BE: Antibiotic-resistant bugs in the 21st century - a clinical super-challenge. N Engl J Med. 2009, 360: 439-443. 10.1056/NEJMp0804651.View ArticlePubMedGoogle Scholar
  2. Tenover FC: Development and spread of bacterial resistance to antimicrobial agents: an overview. Clin Infect Dis. 2001, 33 (Suppl 3): S108-S115. 10.1086/321834.View ArticlePubMedGoogle Scholar
  3. Leavis HL, Willems RJ, van Wamel WJ, Schuren FH, Caspers MP, Bonten MJ: Insertion sequence-driven diversification creates a globally dispersed emerging multiresistant subspecies of E. faecium. PLoS Pathog. 2007, 3: e7-75. 10.1371/journal.ppat.0030007.View ArticlePubMedPubMed CentralGoogle Scholar
  4. Willems RJ, van SW: Transition of Enterococcus faecium from commensal organism to nosocomial pathogen. Future Microbiol. 2009, 4: 1125-1135. 10.2217/fmb.09.82.View ArticlePubMedGoogle Scholar
  5. Lester CH, Sandvang D, Olsen SS, Schonheyder HC, Jarlov JO, Bangsborg J, Hansen DS, Jensen TG, Frimodt-Moller N, Hammerum AM: Emergence of ampicillin-resistant Enterococcus faecium in Danish hospitals. J Antimicrob Chemother. 2008, 62: 1203-1206. 10.1093/jac/dkn360.View ArticlePubMedGoogle Scholar
  6. Top J, Willems R, van d V, Asbroek M, Bonten M: Emergence of clonal complex 17 Enterococcus faecium in The Netherlands. J Clin Microbiol. 2008, 46: 214-219. 10.1128/JCM.01351-07.View ArticlePubMedGoogle Scholar
  7. Nallapareddy SR, Singh KV, Okhuysen PC, Murray BE: A functional collagen adhesin gene, acm, in clinical isolates of Enterococcus faecium correlates with the recent success of this emerging nosocomial pathogen. Infect Immun. 2008, 76: 4110-4119. 10.1128/IAI.00375-08.View ArticlePubMedPubMed CentralGoogle Scholar
  8. Sillanpaa J, Prakash VP, Nallapareddy SR, Murray BE: Distribution of genes encoding MSCRAMMs and pili in clinical and natural populations of Enterococcus faecium. J Clin Microbiol. 2009, 47: 896-901. 10.1128/JCM.02283-08.View ArticlePubMedPubMed CentralGoogle Scholar
  9. Hendrickx APA, Van Wamel WJB, Posthuma G, Bonten MJM, Willems RJL: Five genes encoding surface exposed LPXTG proteins are enriched in hospital-adapted Enterococcus faecium Clonal Complex-17 isolates. J Bacteriol. 2007, 189: 8321-8332. 10.1128/JB.00664-07.View ArticlePubMedPubMed CentralGoogle Scholar
  10. Hendrickx AP, Willems RJ, Bonten MJ, van SW: LPxTG surface proteins of enterococci. Trends Microbiol. 2009, 17: 423-430. 10.1016/j.tim.2009.06.004.View ArticlePubMedGoogle Scholar
  11. Freitas AR, Tedim AP, Novais C, Ruiz-Garbajosa P, Werner G, Laverde-Gomez JA, Canton R, Peixe L, Baquero F, Coque TM: Global spread of colonization-virulence hyl Efm gene in megaplasmids of CC17 Enterococcus faecium polyclonal sub-cluster. Antimicrob Agents Chemother. 2010, 54: 2660-2665. 10.1128/AAC.00134-10.View ArticlePubMedPubMed CentralGoogle Scholar
  12. Rice LB, Carias L, Rudin S, Vael C, Goossens H, Konstabel C, Klare I, Nallapareddy SR, Huang W, Murray BE: A potential virulence gene, hyl Efm, predominates in Enterococcus faecium of clinical origin. J Infect Dis. 2003, 187: 508-512. 10.1086/367711.View ArticlePubMedGoogle Scholar
  13. Heikens E, Bonten MJM, Willems RJL: Enterococcal surface protein Esp is important for biofilm formation of Enterococcus faecium E1162. J Bacteriol. 2007, 189: 8233-8240. 10.1128/JB.01205-07.View ArticlePubMedPubMed CentralGoogle Scholar
  14. Willems RJ, Homan W, Top J, van Santen-Verheuvel M, Tribe D, Manzioros X, Gaillard C, Vandenbroucke-Grauls CM, Mascini EM, van KE, et al: Variant esp gene as a marker of a distinct genetic lineage of vancomycin-resistant Enterococcus faecium spreading in hospitals. Lancet. 2001, 357: 853-855. 10.1016/S0140-6736(00)04205-7.View ArticlePubMedGoogle Scholar
  15. Heikens E, van Schaik W, Leavis HL, Bonten MJM, Willems RJL: Identification of a novel genomic island specific to hospital-acquired clonal complex 17 Enterococcus faecium isolates. Appl Environ Microbiol. 2008, 74: 7094-7097. 10.1128/AEM.01378-08.View ArticlePubMedPubMed CentralGoogle Scholar
  16. Lester CH, Olsen SS, Schonheyder HC, Hansen DS, Tvede M, Holm A, Arpi M, Friis-Moller A, Jensen KT, Kemp M, et al: Typing of vancomycin-resistant enterococci obtained from patients at Danish hospitals and detection of a genomic island specific to CC17 Enterococcus faecium. Int J Antimicrob Agents. 2009, 35: 312-314. 10.1016/j.ijantimicag.2009.11.014.View ArticlePubMedGoogle Scholar
  17. Nallapareddy SR, Weinstock GM, Murray BE: Clinical isolates of Enterococcus faecium exhibit strain-specific collagen binding mediated by Acm, a new member of the MSCRAMM family. Mol Microbiol. 2003, 47: 1733-1747. 10.1046/j.1365-2958.2003.03417.x.View ArticlePubMedGoogle Scholar
  18. Donabedian S, Thal LA, Bozigar P, Zervos T, Hershberger E, Zervos M: Antimicrobial resistance in swine and chickens fed virginiamycin for growth promotion. J Microbiol Methods. 2003, 55: 739-743. 10.1016/j.mimet.2003.07.002.View ArticlePubMedGoogle Scholar
  19. McDonald LC, Rossiter S, Mackinson C, Wang YY, Johnson S, Sullivan M, Sokolow R, DeBess E, Gilbert L, Benson JA, et al: Quinupristin-dalfopristin-resistant Enterococcus faecium on chicken and in human stool specimens. N Engl J Med. 2001, 345: 1155-1160. 10.1056/NEJMoa010805.View ArticlePubMedGoogle Scholar
  20. Jung WK, Lim JY, Kwon NH, Kim JM, Hong SK, Koo HC, Kim SH, Park YH: Vancomycin-resistant enterococci from animal sources in Korea. Int J Food Microbiol. 2007, 113: 102-107. 10.1016/j.ijfoodmicro.2006.07.023.View ArticlePubMedGoogle Scholar
  21. Klare I, Heier H, Claus H, Bohme G, Marin S, Seltmann G, Hakenbeck R, Antanassova V, Witte W: Enterococcus faecium strains with vanA-mediated high-level glycopeptide resistance isolated from animal foodstuffs and fecal samples of humans in the community. Microb Drug Resist. 1995, 1: 265-272. 10.1089/mdr.1995.1.265.View ArticlePubMedGoogle Scholar
  22. Klare I, Heier H, Claus H, Reissbrodt R, Witte W: vanA-mediated high-level glycopeptide resistance in Enterococcus faecium from animal husbandry. FEMS Microbiol Lett. 1995, 125: 165-171. 10.1111/j.1574-6968.1995.tb07353.x.View ArticlePubMedGoogle Scholar
  23. Klare I, Badstubner D, Konstabel C, Bohme G, Claus H, Witte W: Decreased incidence of VanA-type vancomycin-resistant enterococci isolated from poultry meat and from fecal samples of humans in the community after discontinuation of avoparcin usage in animal husbandry. Microb Drug Resist. 1999, 5: 45-52. 10.1089/mdr.1999.5.45.View ArticlePubMedGoogle Scholar
  24. Lietzau S, Hoewner M, von BH, Marre R, Brenner H: Antibiotic resistant fecal isolates of Enterococci among unselected patients outside the clinical sector: an epidemiological study from Southern Germany. Pharmacoepidemiol Drug Saf. 2006, 15: 275-277. 10.1002/pds.1167.View ArticlePubMedGoogle Scholar
  25. Witte W, Werner G, Tietze E, Reissbrodt R, Klare I: Routes of transmission of antibiotic-resistant bacteria and their resistance determinants to humans via various environmental sources like crop food or bathing freshwater. Final report projekt UFOPLAN. 20162211, 2001 - 2004. 1-95. 1-3-2005
  26. Werner G, Klare I, Witte W: Large conjugative vanA plasmids in vancomycin-resistant Enterococcus faecium. J Clin Microbiol. 1999, 37: 2383-2384.PubMedPubMed CentralGoogle Scholar
  27. Klare I, Konstabel C, Mueller-Bertling S, Werner G, Strommenger B, Kettlitz C, Borgmann S, Schulte B, Jonas D, Serr A, et al: Spread of ampicillin/vancomycin-resistant Enterococcus faecium of the epidemic-virulent clonal complex-17 carrying the genes esp and hyl in German hospitals. Eur J Clin Microbiol Infect Dis. 2005, 24: 815-825. 10.1007/s10096-005-0056-0.View ArticlePubMedGoogle Scholar
  28. Freitas AR, Novais C, Ruiz-Garbajosa P, Coque TM, Peixe L: Clonal expansion within clonal complex 2 and spread of vancomycin-resistant plasmids among different genetic lineages of Enterococcus faecalis from Portugal. J Antimicrob Chemother. 2009, 63: 1104-1111. 10.1093/jac/dkp103.View ArticlePubMedGoogle Scholar
  29. Feil EJ, Li BC, Aanensen DM, Hanage WP, Spratt BG: eBURST: inferring patterns of evolutionary descent among clusters of related bacterial genotypes from multilocus sequence typing data. J Bacteriol. 2004, 186: 1518-1530. 10.1128/JB.186.5.1518-1530.2004.View ArticlePubMedPubMed CentralGoogle Scholar
  30. Francisco A, Bugalho M, Ramirez M, Carrico J: Global optimal eBURST analysis of multilocus typing data using a graphic matroid approach. BMC Bioinformatics. 2009, 10: 152-10.1186/1471-2105-10-152.View ArticlePubMedPubMed CentralGoogle Scholar
  31. Grady R, Hayes F: Axe-Txe, a broad-spectrum proteic toxin-antitoxin system specified by a multidrug-resistant, clinical isolate of Enterococcus faecium. Mol Microbiol. 2003, 47: 1419-1432. 10.1046/j.1365-2958.2003.03387.x.View ArticlePubMedGoogle Scholar
  32. Dahl KH, Rokenes TP, Lundblad EW, Sundsfjord A: Nonconjugative transposition of the vanB-containing Tn5382-like element in Enterococcus faecium. Antimicrob Agents Chemother. 2003, 47: 786-789. 10.1128/AAC.47.2.786-789.2003.View ArticlePubMedPubMed CentralGoogle Scholar
  33. van Schaik W, Top J, Riley DR, Boekhorst J, Vrijenhoek JEPV, Schapendonk CME, Hendrickx APA, Nijman IJ, Bonten MJM, Tettelin H, et al: Pyrosequencing-based comparative genome analysis of the nosocomial pathogen Enterococcus faecium and identification of a large transferable pathogenicity island. BMC Genomics. 2010, 11: 239-10.1186/1471-2164-11-239. BMC Genomics 2010View ArticlePubMedPubMed CentralGoogle Scholar
  34. Galloway-Pena JR, Nallapareddy SR, Arias CA, Eliopoulos GM, Murray BE: Analysis of clonality and antibiotic resistance among early clinical isolates of Enterococcus faecium in the United States. J Infect Dis. 2009, 15: 1566-1573. 10.1086/644790.View ArticleGoogle Scholar
  35. Leavis HL, Willems RJL, Top J, Bonten MJM: High-Level ciprofloxacin resistance from point mutations in gyrA and parC confined to global hospital-adapted clonal lineage CC17 of Enterococcus faecium. J Clin Microbiol. 2006, 44: 1059-1064. 10.1128/JCM.44.3.1059-1064.2006.View ArticlePubMedPubMed CentralGoogle Scholar
  36. Werner G, Fleige C, Ewert B, Laverde-Gomez JA, Klare I, Witte W: High-level ciprofloxacin resistance among hospital-adapted Enterococcus faecium (CC17). Int J Antimicrob Agents. 2010, 35: 119-125. 10.1016/j.ijantimicag.2009.10.012.View ArticlePubMedGoogle Scholar
  37. Werner G, Coque TM, Hammerum AM, Hope R, Hryniewicz W, Johnson A, Klare I, Kristinsson KG, Leclercq R, Lester CH, et al: Emergence and spread of vancomycin resistance among enterococci in Europe. Euro Surveill. 2008, 13:Google Scholar
  38. Simjee S, McDermott PF, White DG, Hofacre C, Berghaus RD, Carter PJ, Stewart L, Liu T, Maier M, Maurer JJ: Antimicrobial susceptibility and distribution of antimicrobial-resistance genes among Enterococcus and coagulase-negative Staphylococcus isolates recovered from poultry litter. Avian Dis. 2007, 51: 884-892. 10.1637/7973-032607-REGR.1.View ArticlePubMedGoogle Scholar
  39. Top J, Willems R, Bonten M: Emergence of CC17 Enterococcus faecium: from commensal to hospital-adapted pathogen. FEMS Immunol Med Microbiol. 2008, 52: 297-308. 10.1111/j.1574-695X.2008.00383.x.View ArticlePubMedGoogle Scholar
  40. Willems RJ, Bonten MJ: Glycopeptide-resistant enterococci: deciphering virulence, resistance and epidemicity. Curr Opin Inf Dis. 2007, 20: 384-390. 10.1097/QCO.0b013e32818be63d.View ArticleGoogle Scholar
  41. Freitas AR, Novais C, Ruiz-Garbajosa P, Coque TM, Peixe L: Dispersion of multidrug-resistant Enterococcus faecium isolates belonging to major clonal complexes in different Portuguese settings. Appl Environ Microbiol. 2009, 75: 4904-4908. 10.1128/AEM.02945-08.View ArticlePubMedPubMed CentralGoogle Scholar
  42. Arias CA, Panesso D, Singh KV, Rice LB, Murray BE: Co-Transfer of antibiotic resistance genes and a hyl(Efm)-containing virulence plasmid in Enterococcus faecium. Antimicrob Agents Chemother. 2009, 53: 4240-4246. 10.1128/AAC.00242-09.View ArticlePubMedPubMed CentralGoogle Scholar
  43. Laverde-Gomez JA, van Schaik W, Freitas AR, Coque TM, Weaver K, Francia MV, Witte W, Werner G: A multiresistance megaplasmid bearing a hyl Efm genomic island in hospital Enterococcus faecium isolates. Int J Med Microbiol. 2011, 301: 165-175. 10.1016/j.ijmm.2010.08.015.View ArticlePubMedGoogle Scholar
  44. Burrus V, Waldor MK: Shaping bacterial genomes with integrative and conjugative elements. Research in Microbiology. 2004, 155: 376-386. 10.1016/j.resmic.2004.01.012.View ArticlePubMedGoogle Scholar
  45. Pre-publication history

    1. The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/11/80/prepub

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