Outbreak detection, screening and infection control measures
In the 1500-bed University Hospital Muenster, routine surveillance, i.e. regular review of patients’ charts and microbiological test results, detected five VRE infections on the hematologic/oncologic ward between January and April 2015. In addition 12 VRE colonisations could be detected coincidentally in anal swabs or stool samples in epidemiologically linked patients. As these rates exceeded the baseline of two infections and three incidentally detected colonisations every 12 months, an outbreak investigation was initiated. Subsequently, VRE point prevalence among all patients on ward was determined and environmental samples were taken. A VRE infection control strategy (hereafter called “VRE bundle strategy”) was established including the following measures: Patients were screened upon admission and contact precautions were implemented. Patients with positive VRE testing were isolated. Isolation of more than one patient in one room was performed if patients were colonized or infected with enterococci harbouring identical vanA/B resistance genes. Separation of toilets, showers, and water supplies was performed and previously shared bathing rooms were closed for colonised patients from this moment on. Staff was instructed to wear personal protective equipment in case of entering a patient room, consisting of gloves, surgical masks and gowns. Surface disinfection was performed initially in every room, including washrooms, patient rooms, nurses’ room, storage rooms, and staff rest rooms once a day using Perform® (Schülke & Mayr GmbH, Norderstedt, Germany). Hand hygiene training was performed among nurses, physicians, cleaning personnel, and kitchen staff. Implementation of hygienic measures was observed by infection control staff every day. A time line was compiled, documenting every patient’s VRE status on the ward. Patients with known VRE colonisations, detected during previous hospital stays, were immediately isolated in a single patient room. De-isolation was only performed in case of three negative swab samples collected in three consecutive weeks without application of any antibiotics within this period.
After an additional VRE infection (sepsis) in August 2015, a weekly screening was added to the VRE bundle strategy in order to clearly identify hospital-acquired colonisations and infections. Transmissions were classified as nosocomial colonisations or infections if they occurred >48 h after hospitalization and the initial screening was negative or not performed.
VRE screening, culture and PCR testing methods
VRE screening was performed obtaining rectal (5 cm ab ano) swabs (Transwab ® m40 compliant, mwe, Corsham, Wiltshire, UK) that were applied to blood agar (Columbia sheep blood agar, Oxoid, Wesel, Germany) and a chromogenic selective agar (VRESelect™, Biorad, Hercules, California, USA) and incubated for up to 48 h at 37 °C. Bacterial species of suspected colonies were confirmed by MALDI-TOF-MS (Bruker Corporation, Bremen, Germany) and antibiotic susceptibility testing was performed and verified using VITEK®2 system (BioMérieux, Nürtingen, Germany) in accordance with the EUCAST standards for clinical breakpoints. In case of vancomycin resistance, the GenoType Enterococcus system (Hain Lifescience, Nehren, Germany) was used to differentiate vancomycin resistance genes vanA, vanB, vanC1 and vanC2/C3.
Environmental sampling and testing methods
Two series of environmental sampling were performed: first during the initial phase after transmission detection in May 2015, second after cleaning of hand contact surfaces in June 2015. Polywipes (mwe, Corsham, Wiltshire, UK) were applied on surfaces and incubated in Tryptic Soy Broth + LT (Merck Millipore, Eppelheim, Germany) for 24 h at 37 °C. Following, 10 μL of broth were applied to blood agar and VRE selective agar and incubated for 24 h at 37 °C. Suspected colonies were subcultured on blood agar and species identification was performed with the help of MALDI-TOF-MS (Bruker Corporation). Susceptibility testing for vancomycin was performed using Etest® (Bestbion GmbH, Liofilchem, Italy) and evaluated in accordance with the EUCAST standards for clinical breakpoints.
Whole genome sequence-based typing
To determine the clonal relationship of isolated VRE strains, the isolates were subjected to whole genome sequencing (WGS) using the Illumina MiSeq platform (Illumina Inc., San Diego, USA) as described previously [15]. After sequencing, quality-trimming and de novo assembly were performed, coding regions were compared in a gene-by-gene approach (core genome Multilocus SequenceTyping, cgMLST) [16] using the SeqSphere+ software version 2.0 beta (Ridom GmbH, Muenster, Germany). The clonal relationship was displayed in a minimum-spanning tree that was generated using the same software. For backwards compatibility with classical molecular typing, i. e. MLST, the MLST sequence types (ST) were extracted from the WGS data in silico.
Statistical analysis
All data are expressed as absolute numbers or percentage, if not stated otherwise. Statistical analyses were performed using the Fisher’s exact test for categorical data. Statistical significance was declared at p < 0.05.