ebd environmental dna and infections 1131
in nosocomial pathogens. Such material, previously con- sidered clinically inconsequential or inert, may be present in viable-yet-uncultivable bacteria, in dead cells, or even in extracellular (ie, naked) form.13 Recent observations that pathogenic bacteria can integrate short, damaged DNA frag- ments into their chromosomes expand the potential implica- tions of this contaminant.13 Our goal was to assess the relationships among environmental
DNA, cultivable target organisms (TOs), cleaning, and HAI in a newly opened EBD hospital. DNA included both general (non–species-specific) 16S bacterial DNA, and DNA of species- specific TOs (listed in the Methods section). Because those taxa referred to as the ESKAPE pathogens (ie, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa,and Enterobacter spp.) are among the most nosocomially relevant, we focused on members of that group, as well as E. coli and Clostridium difficile.Further- more, financial and technical constraints precluded us from studying viral, fungal, and anaerobic organisms.We also sought to prospectively determine: (1) patterns of environmental contamination beginning before the facility opened and after routine terminal cleaning; (2) the genetic relatedness, based on whole-genome sequencing (WGS), of any multidrug-resistant target organism (MDR-TO) isolated from the environment to those isolated from HAIs; and (3) the correlation between environmental TO contamination levels and the frequency of analogous HAIs.
methods Setting
Fort Belvoir Community Hospital (FBCH) is a newly con- structed 120-bed, EBD facility in northeastern Virginia. It opened in September 2011 and has 10 operating rooms, a medical-surgical intensive care unit, a telemetry unit, separate medical and surgical wards, and pediatrics and maternity wards. Some bed rail and mattress surfaces (those on the long-term, anti-pressure ulcer beds) in the intensive care unit and telemetry unit are copper impregnated. Although the public restroom faucets are touch-free, all others in the facility are not. FBHC has no ultraviolet units or fogging systems for enhanced room/ surface decontamination. FBHC has a beneficiary population exceeding 90,000 and supports one of the busiest emergency departments in the northern capital region. At FBHC in 2014, there were 9,340 inpatient surgeries, 48,793 emergency depart- ment patients, 24,976 inpatient bed days, 680,000 visits to ambulatory care clinics, and 1,569 births. The average daily census was 73.
Definitions
We defined thorough cleaning as whether a surface had been wiped enough to remove at least 90% of an invisible marking dye (DAZO fluorescent marking gel, Kleancheck Systems, Hingham, MA) that fluoresces under ultraviolet light. Effective
cleaning was defined as surface with no detectable biomaterial. Biomaterial was defined as any microbial growth on blood or MacConkey culture media, or a detectable signal from real-time polymerase chain reaction (PCR) indicating the presence of general or species-specific bacterial DNA. TOs included any of the following: Acinetobacter baumannii; A. baumannii calcoaceticus complex;
E.coli;
P.aeruginosa;S. aureus; K. pneumoniae; E. faecium (or faecalis); E. cloacae (or aerogenes); and C. difficile. Multidrug resistance (MDR) was defined according toMagiorakos et al.14 Inpatient infection or acquisition was defined as no evidence of infection or a negative surveillance culture upon admission, followed by a positive clinical or surveillance culture arising no less than 48 hours after admission.
Terminal Cleaning and Surveillance
Terminal cleaning was performed in a standard manner involving collection of trash and linen, high dusting, wet dusting, wiping of surfaces with a quaternary ammonium compound disinfectant, floor cleaning, bathroom cleaning, and room inspection. Surveillance was prospectively con- ducted for 16 months from October 2011 through January 2013. After patients were discharged, but prior to terminal cleaning of their rooms, 17 high-touch surfaces were sampled for 20 seconds using rayon-tipped swabs premoistened with nutrient transport media. Surfaces were then marked with DAZO gel. After terminal cleaning, the presence or absence of the dye was assessed using the EnCompass monitoring system (Ecolab, St. Paul, MN). Surfaces were then resampled as described above. If the evaluation of the cleaned room could not occur shortly after terminal cleaning, the surveillance episode for that room was aborted. Swabs were used instead of sponges for 2 reasons: (1) swabs are more sensitive than sponges for detecting certain, particularly problematic TOs (eg, Acinetobacter) in the hospital environment15; and (2) sponges were too large to fit in tubes used to extract template for PCR. Each surface was sampled with a separate swab.
Sample Processing Culture method. Swabs were immediately transported to a
processing lab and streaked in seriatim onto a blood agar plate (BAP) and a MacConkey agar (MAC) plate. BAP and MAC plates were incubated for 24–48 hours at 35°C. Gram-positive organisms growing on BAP were taxonomically identified using rapid tests (coagulase, catalase, and Staphaurex Remel, Thermo Scientific, Waltham, MA) followed by analysis on the Phoenix automated system (Bekton Dickenson, Franklin Lakes,NJ) using the PMIC/ID-107 panel. Colonies that grew on MAC plates (which were presumably Gram negative) were analyzed on the Phoenix using panel NMIC/ID-133. Any potential TO not definitively identifiable by these methods was further analyzed using a matrix-assisted laser desorption ionization mass spectrometer (MALDI Biotyper, Bruker, Billerica, MA). PCR methods. After streaking onto BAP and MAC media, swab tips were aseptically removed from rods, submerged in
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