Setting
The Tel Aviv Sourasky Medical Center (TASMC) NICU consists of a total of 49 beds, of which 22 beds comprise a level-IIIB NICU and 27 beds comprise a level-IIA NICU. In addition, there is a special-care nursery with the capacity of 13 beds which is staffed and administered in a facility separate from the NICU. Sick or at-risk neonates are admitted directly after birth into either the NICU or the special-care nursery, depending upon the severity of their illness. They are moved between units as their needs change.
Patients
The index case was a newborn that needed cardiac surgery and who was identified by positive screening for MDROs upon arrival to another hospital’s NICU.
Outbreak management and intervention
After the first case of VRE-fm infection was detected, all of the hospitalized neonates were enrolled in active surveillance for VRE colonization according to the following steps:
- 1.
All neonates in the TASMC NICU were immediately screened for VRE-fm.
- 2.
The hospital management and the Ministry of Health (MoH) were informed of a VRE outbreak.
- 3.
A designated VRE group was isolated in the NICU. Since over half of the infants screened positive for VRE-fm, after consulting with the MoH, it was decided that positive and exposed infants (i.e., all infants in the NICU at this time point) will comprise a single cohort and be treated according to the same protocol. This group was separated from all new NICU admissions. The movement of neonates within and between units was restricted, and the entrance of outside staff into the units was kept to a minimum.
- 4.
One room was designated for new NICU admissions and defined as “the clean area”. This room was meticulously cleaned prior to the first new admission. Disposable supplies were either destroyed or moved to the VRE area.
- 5.
Physicians, nurses and nurses’ aides were assigned to work exclusively either in the designated VRE area or in the clean area.
- 6.
A special protocol was followed for cases requiring a specific physician to treat a patient from the VRE group. The protocol required the use of gloves and protective yellow clothing before entering the VRE area and changing into regular hospital gear before leaving the VRE area. Health professionals (e.g., x-ray technicians) who needed to see patients in both groups were instructed to begin in the clean area and to follow the above protocol in the VRE area.
- 7.
All parents were informed by the attending physicians in the NICU about the outbreak, and provided with a written explanation. Information was provided to parents and visitors about the requirement of hand hygiene and the reasons for the enhanced infection control measures for VRE-fm colonized babies.
- 8.
A separate entrance, an area for breastfeeding, and a waiting area were designated for families of infants in the VRE group. These families were given special instruction on the importance of hand hygiene and were asked not to enter other areas of the hospital.
- 9.
The education of newborn service and visiting hospital staff now centered around hand hygiene and on the potential role of healthcare workers in the transmission of VRE-fm.
- 10.
The rates of adherence to the WHO guideline on Hand Hygiene [15] were monitored and included in the feedback to the units throughout the outbreak period.
Sample collection, screening and antibiotic susceptibility evaluation
Stool swabs were collected from patients, inoculated on CHROMagar VRE™ plates (Hylabs, Rehovot, Israel) and incubated at 36 °C for 18–24 h. Growth of colonies was detected according to the manufacturer’s instructions, and colony species were identified by the VITEK-MS® system (bioMérieux SA, Marcy l’Etoile, France). Vancomycin resistance and antibiotic susceptibility were determined by VITEK® 2 (bioMérieux SA, Marcy l’Etoile, France).
Environmental investigation
Environmental sampling was performed in an attempt to identify a possible environmental source for the outbreak. In total, 10 environmental sites were sampled with premoistened wipes (Polywipe®, Medical Wire and Equipment, Wiltshire, UK), including sinks, taps, and incubators. Furthermore, the samples collected in the NICU included handles of hand disinfection dispensers, soap dispensers, and a variety of environmental surfaces. The wipes were inoculated on CHROMagar VRE™ plates (Hylabs, Rehovot, Israel) and then incubated in a brain heart infusion broth (BHI, Hylabs, Rehovot, Israel). Identification of suspected colonies and analysis of antibiotic resistance were performed as described above. VRE-confirmed colonies were tested for the presence of vancomycin-resistant genes, and clonality was determined by molecular genotyping.
Detection of the vanA/vanB vancomycin resistance genes
PCR amplification was performed for detection of the genes as previously described [16] using the primers vanA F: 5′-GGGAAAACGACAATTGC-3′, vanA R: 5′-GTACAATGCGGCCGTTA-3′, and vanB F: 5′-ACGGAATGGGAAGCCGA-3′, vanB R: 5′-TGCACCCGATTTCGTTC-3′ with 68 °C annealing temperature and 35 cycles. E. faecium 5842 and E. faecalis 3528 harboring vanA and vanB, respectively, were used as positive controls.
Molecular genotyping
Genotyping of VRE isolates was performed using by BOX-PCR. Prior to analysis, each VRE isolate was streaked onto a Mueller-Hinton (MH) agar plate (Hylabs, Rehovot, Israel) and incubated at 37 °C. BOX-PCR was performed as previously described [17] with the following modifications. 100 ng of DNA extracted from one colony from each plate was suspended in 7.5 μl sterile ddH2O and added to 10 μl of PCR mastermix (OneTaq®, NEB, UK). 0.5 μl (50 μM) BOX A2R primer was added to obtain a final volume of 18 μl. Samples were denatured at 95 °C for 7 min, amplified in 35 cycles of 90 °C for 30 s, 40 °C for 1 min, and 68 °C for 8 min, followed by a final extension step of 68 °C for 16 min. Banding patterns were visualized using QIAxcel ScreenGel software (Qiagen, USA) combined with the QIAxcel Advanced capillary electrophoresis system (Qiagen, USA). A E. faecium 5842 reference strain was included as a discriminatory control. Fingerprint patterns of the isolates generated by BOX-PCR were analyzed with GelCompar II software (Applied Maths, Belgium). Dendrograms were created using a densitometric curve-based algorithm (Dice correlation coefficient) and UPGMA to cluster patterns by similarity.