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 [18–20]. 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.

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, 39–41]. 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].

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].