The neonatal ICU belongs to a hospital of tertiary care (2.300 beds) located in South-Western Germany. It consists actually of two units with one containing 16 beds in four rooms and an additional one containing 20 beds in 5 rooms. Unit 1 offers the possibility of mechanical ventilation; patients are regularly transferred between the two units. The setting is conceptually a mixed ward allowing also older, pediatric patients to be admitted into two of the rooms. The neonatal ICU is a so-called “Perineonatology level 1 Centre” with an annual number of about 50 neonatal patients with a low birth weight of <1.500 g.
Patient setting, bacterial isolates and primary diagnostics
During a period of September 2008 until June 2009 altogether 598 patients attending a neonatal ICU were screened non-selectively for enterococci. Columbia Agar with sheep blood (COL SB; Oxoid, Wesel, Germany) was used to isolate enterococci from different clinical samples. E. faecium was identified by using standard microbiological methods including hydrolyzing esculin and growth in 6.5% NaCl and by API 20 Strep (bioMérieux, Nürtingen, Germany). Randomly chosen enterococcal isolates were subsequently tested for resistance to vancomycin by routine diagnostics using agar diffusion or Etest® Vancomycin (bioMérieux). Susceptibility interpretations followed the guidelines proposed by CLSI (S ≤4; I =8/16; R ≥32 mg/L). Vancomycin resistance genotypes (vanA, vanB or vanC) were determined by a PCR and Southern hybridization based assay (GenoType® Enterococcus, Hain Lifescience, Nehren, Germany). Discrepancies between phenotypic (susceptible) and genotypic (vanB-positive) results lead to the general agreement to test all isolates for vanB with an inhibition zone of ≤18 mm around a vancomycin disk by a genotypic method. Seventy-one pre-selected E. faecium isolates were sent for vanB type confirmation and clonal analysis to the German focal laboratory for enterococci at the Robert Koch Institute.
Antibiotic susceptibility testing
For all 71 E. faecium isolates antibiotic susceptibilities were determined for 14 antibiotics as minimal inhibitory concentrations (MIC) using a microdilution method in cation-adjusted Mueller-Hinton broth according to international standards. We used the EUCAST clinical breakpoints when available; for other antibiotics we applied breakpoints derived from CLSI, DIN and based on other criteria (e.g., for high level ciprofloxacin resistance >16 mg/L ). MICs were classified as resistant (in mg/L) as follows: penicillin/ampicillin >8, vancomycin >4; teicoplanin >2, erythromycin >4, linezolid >4, tetracycline >4, rifampicin >0.5, chloramphenicol >16, tigecycline >0.5, daptomycin >4, gentamicin (high-level) >128, streptomycin (high-level) >512, quinupristin/dalfopristin >4. Etest for vancomycin was performed according to the recommendation of the manufacturer (bioMérieux). In brief, two different protocols were followed. First, a standard screening method with Mueller-Hinton agar and an inoculum equivalent to McFarland 0.5 and second, Brain Heart Infusion agar and an inoculum equivalent to McFarland 2.0 was used. The latter one is called Etest® macromethod and is suggested for a confirmation of a supposed vancomycin resistance phenotype. Values are read after incubation at 35°C for 24 and 48 h as recommended (Etest® application sheet for Enterococcus/VRE and vancomycin EAS009). E. faecalis ATCC29212 and E. faecium ATCC19434 were used as control strains. Performance of three commercially available, chromogenic VRE screening agars was evaluated; Oxoid BrillianceTM agar VRE (Thermo Scientific Fisher, Wesel, Germany); chromIDTM VRE (bioMérieux) and CHROMagarTM VRE (Mast Diagnostika, Reinfeld, Germany). Strains were streaked out on selective plates and incubated as recommended by the manufacturers. Growth as single colonies and with the equivalent colours was rated as a positive result.
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. Plasmids were extracted according to an alkaline lysis protocol and subsequent phenol/chloroform-based purification as described recently .
PCR was performed with a PCR master mix (Thermo Fisher Scientific; St. Leon-Rot, Germany) according to the manufacturer’s instructions. Exactly 0.5 μL of isolated genomic DNA (ca. 10 ng) and primers (200 nM each) were added. Amplification of fragments representing the esp
and vanA/B genes was performed in a multiplex PCR as described elsewhere . Subtyping of vanB ligases and cluster types was done as described recently [13, 14]. Primers vanB-L1: 5’-GTTTGATGCAGAGGCAGACGACT and vanB-L2 5’- ACAAGTTCCCCTGTATCCAAGTGG were used to amplify a 5,959 bp product using the Expand Long Template PCR system and conditions set by the manufacturer (Roche Applied Science, Mannheim, Germany). Long PCR products were subsequently digested with BspH1 and DraI for 2 h at 37°C and resolved in 0.8% agarose gels. Plasmid replicase genes were amplified as described [15, 16]. PCR for IS16 was performed as described . The following strains and plasmids were used as positive control samples: plasmid pRUM (IS16, rep
family), plasmid pLG1 (hyl
, repA-N family, new subtype), plasmid pIP816 (vanA; E. faecium BM4147), E. faecium U0317 (esp), and E. faecalis V583 (vanB) and E. faecalis RE25 pRE25 (inc18 rep2 family). E. faecalis OG1RF served as a negative control sample for all PCR assays.
Altogether nine ST192 strains (UW7606, UW7609, UW7611, UW7612, UW7813, UW7819, UW7835, UW7842, UW7845) were used as donors in in vitro filter-mating experiments. The rifampicin- and fusidic acid-resistant E. faecium strain 64/3 was used as a recipient. Transconjugants were selected on BHI agar supplemented with rifampicin (30 mg/L), fusidic acid (20 mg/L) and vancomycin with various concentrations according to the MIC of the donor strain (2, 4, 8 mg/L). The mating protocol was performed and mating rates were calculated as described recently . Plates were incubated at 37°C up to 48 h. Supposed transconjugants were grown on selective plates and analyzed phenotypically (antibiotic susceptibilities) and genotypically (PCR-based marker genes and PFGE).
Genomic DNA for PFGE analysis was isolated and treated as described recently . The agarose gel concentration was 1%, the CHEF-DR III apparatus (Bio-Rad Laboratories, Hercules, CA, USA) was used for PFGE. SmaI-digested Staphylococcus aureus NCTC 8325 was used as a molecular mass standard on all PFGE gels. Genomic DNA of the E. faecium isolates was digested with SmaI. The ramped pulsed times were as follows: 1 – 11 s for 15 h and 11 – 30 s for 14 h at 14°C. Digestion of genomic DNA with I-Ceu-I linearises chromosomal DNA by recognizing the six rDNA operons in E. faecium revealing six chromosomal bands in PFGE. Genomic DNA was digested with I-Ceu-I for 16 h at 37°C. The ramped pulsed times for I-Ceu-I gels were 5 – 30 s for 22 h at 14°C .
Southern hybridization experiments were done as described elsewhere using a PCR-generated digoxigenin-labelled vanB probe (DIG High Prime; Roche Applied Science), hybridization chemicals and equipment from commercial kits and according to recommendations of the manufacturer (Roche Applied Science). Immunological detection was done as recommended using a chemiluminescent probe (CDP-StarTM, Roche Applied Science) 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 (Life Technologies/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/).
Statistical analyses were performed with software package EpiCompare 1.0 (Ridom).