CTA collection

Unused CTAs were collected in Ziploc-style specimen bags directly from a box on 3 different dates prior to patient use. After use, CTAs were collected in the same type of bags during a 2- month period from a convenience sample of patient rooms in a 28-bed medical intensive care unit immediately after patient discharge but prior to room cleaning. All CTAs were cultured the same afternoon, immediately after transport to the research laboratory. We did not record the length of stay or other clinical information for patients occupying rooms from which the CTAs were removed. In total, 17 CTAs were cultured directly out of the box and 23 were cultured after patient use.

Bacterial culture and identification

All studies were performed in a research laboratory that routinely performs bacterial cultures. For each CTA, a sterile swab was dipped in sterile phosphate-buffered saline (PBS) and wiped over the entire outside surface. The swab contents were inoculated directly to a Lysogeny Broth (LB) agar plate, which was incubated at 37 °C for 48 hours. The negative control consisted of 100 µL PBS inoculated to a LB agar plate under similar conditions. Colony-forming units (CFU) were recorded, and bacteria of similar phenotypes were picked and transferred to 5% sheep blood agar plates (Remel, Lenexa, KS) for identification by the Yale New Haven Hospital Clinical Microbiology Laboratory using the Vitek-2 matrix-assisted laser desorption/ionization time of flight mass spectrometer (MALDI-TOF MS).

Low-level disinfection of CTAs

Next, we evaluated the ability of 2 different disinfectant wipes, 0.5% hydrogen peroxide (Oxivir1) or 0.25% tetra-ammonium chloride with 44.50% isopropyl alcohol (Super Sani-Cloth), to remove bacteria from the CTAs. Both products are registered by the US Environmental Protection Agency (EPA) to be effective against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus spp.⁵ The CTAs were inoculated from a suspension of overnight growth of 1 of the following environmental organisms recovered from CTAs from patient rooms: Micrococcus luteus, Staphylococcus epidermidis or Bacillus megaterium. Additionally, we tested 2 pathogenic respiratory clinical isolates: S. aureus and Pseudomonas aeruginosa.

To estimate the initial bacterial inoculum, the overnight suspension of each bacteria was serially diluted in LB, inoculated to an LB agar plate, and incubated at 37 °C overnight. The CFU count was then recorded. Next, 30 µL of each overnight bacterial suspension was added to the CTA surface. The inoculum was spread with a sterile swab and dried for 15 minutes. The B. megaterium grew poorly and was susceptible to desiccation, and fewer bacteria were recovered after 15 minutes of drying. Therefore, for B. megaterium, the CTAs were inoculated with a sterile swab dipped directly in the overnight suspension and dried for 7 minutes instead of 15 minutes. In all cases, no visible liquid was present prior to reculturing the CTA surface.

We then estimated how many bacteria remained after drying, prior to disinfection. The top half of the dried, contaminated CTA was wiped with a sterile cotton swab dipped in LB. The swab was then cut and submerged in a microcentrifuge tube with 1.0 mL LB and vortexed for 30 seconds to release bacteria from the swab. The vortexed LB was serially diluted, the dilutions were inoculated to LB agar plates incubated at 37 °C overnight, and the colonies were then counted. Alternatively, the contaminated surface was wiped directedly with a sterile swab dipped in LB and the swab used to inoculate an LB plate. Most often, this latter method was semiquantitative, producing a lawn of bacteria that confirmed a minimum inoculum of at least >10³ (data not shown).

The CTAs were then disinfected with either 0.5% hydrogen peroxide or 0.25% tetra-ammonium chloride with 44.50% isopropyl alcohol wipes by applying the wipes for 5–10 seconds, followed by a contact time determined by the manufacturers’ instructions (ie, 1 minute for hydrogen peroxide or 2 minutes for tetra-ammonium chloride/isopropyl alcohol).^3,4 The CTAs were then dried for a total of 15 minutes prior to reswabbing for bacteria. For B. megaterium, this duration was reduced to 7 minutes because of the previously described death during drying. The bottom half of the CTA was then recultured with a sterile swab dipped in Dey/Engley (D/E) neutralizing broth (Hardy Diagnostics, Santa Maria, CA) in the area that was not swabbed prior to disinfection. This swab was used to inoculate D/E agar plates that were incubated at 37 °C as described for the previous procedure. Bacterial growth was recorded before and after disinfection for each method from the same CTA. All experiments were performed in triplicate on 3 separate CTAs for each bacterial strain for all conditions tested.