Disinfection of Acinetobacter baumannii-Contaminated Surfaces Relevant to Medical Treatment Facilities with Ultraviolet C Light

Military Medicine, Nov 2007 by Rastogi, Vipin K, Wallace, Lalena, Smith, Lisa S

The efficacy of ultraviolet C (UVC) light (100-280 run) in the decontamination of three hospital-related surfaces, namely, unpainted/painted aluminum (bed railings), stainless steel (operating tables), and scrubs (laboratory coats), was investigated. Acinetobacter baumannii cells were inoculated (10^sup 5^ or 10^sup 3^ cells) on small coupons and dried overnight in a class II biosafety cabinet. Drying resulted in ≤50% loss of viability. The UVC fluence of 90 J/m^sup 2^ was observed to be very effective in the decontamination of cells from all metal coupon surfaces (complete killing). However, the same fluence was ineffective in the decontamination of scrubs. The effectiveness of two other common disinfection practices, that is, 15 minutes of boiling or spraying with 70% ethanol, was investigated for the scrubs. Although ethanol treatment was ineffective, the boiling treatment was very effective (complete killing). These results establish that metal surfaces can be decontaminated with UVC irradiation and boiling treatment is effective for scrub decontamination.

Introduction

The emergence and rapid spread of multidrug-resistant isolates causing nosocomial infections have been of alarming concern in recent years.J An increasing number of Acinetobacter baumannii bloodstream infections in patients at military medical facilities, especially intensive care units, have been reported. Because this bacterium has developed resistance to antimicrobial agents, infection control and disinfection of medical treatment facilities are paramount in ensuring low Acinetobacterrelated mortality rates. Although infected patients are often the sources of A. baumannii infections in health care settings, the ability of this organism to survive for extended periods on environmental surfaces is likely a major contributor to protracted outbreaks in medical treatment facilities.2

A. baumannii is a pleomorphic, aerobic, Gram-negative bacterium (similar in appearance to Haemophilus influenzae) commonly found in water and soil. It generally is a colonizer of low virulence, but it is capable of causing infections. The bacterium commonly colonizes skin, oropharyngeal secretions, respiratory secretions, and urine; it uncommonly colonizes the gastrointestinal tract and is associated with nosocomial pneumonia, bacteremia, and wound infections. Therefore, A. baumanniils commonly referred to as a "hospital opportunist." In one study, the length of survival of a number of Gram-negative bacteria, including Serraifa marcescens, Proteus mkabilis, Acinetobacter spp., Klebsiella pneumoniae, and Enterobacter spp., on seven common fabrics and plastics was investigated.3 The survival times of bacteria ranged from 1 hour to 8 days at a low inoculation level (~ 10^sup 2^ cells per swatch). At a higher inoculation level (~ 10^sup 5^ cells per swatch), however, the survival times ranged from 2 hours to 60 days. On the basis of these findings, careful disinfection and conscientious contact control procedures in medical treatment facilities are clearly important.

Ultraviolet C (UVC) irradiation (100-280 ran) is most commonly associated with the disinfection of liquids, particularly water. However, there has been an interest in its application for disinfection of surfaces and foods. Ultraviolet germicidal irradiation is a recognized method of inactivating a wide range of biological agents.4 The efficacy of ultraviolet irradiation is a function of many different locational and operational factors, including intensity, exposure time, lamp placement, and air movement patterns.56 Microorganisms are particularly vulnerable to UVC light of ~254-nm wavelength, because DNA maximally absorbs ultraviolet light in this region, resulting in formation of thymine dimers and other lethal photoproducts.7

Methods

Bacterial Strain, Culture, and Viability

A culture of A. baumannvi (Walter Reed Army Medical Center no. 652) was procured from Walter Reed Army Medical Center. The culture was streaked for single colonies on tryptic soy agar plates and was incubated at 37°C for 24 hours. A single colony was inoculated into 10 mL of tryptic soy broth and cultured at 37°C for 24 hours. Several glycerol stocks were prepared from this culture and frozen at -80°C.

Using a vial of frozen glycerol stock, a tryptic soy agar plate was streaked for single colonies. A flask containing 500 mL of tryptic soy broth was inoculated with a single colony and cultured at 37°C for 24 hours. The cells were concentrated by centrifugation (5,000 rpm for 15 minutes at 4°C) and resuspended in 0.1 volume of dilute (0.5%) buffered peptone water (BPW). The cells were washed twice in BPW, suspended in 20 mL of BPW, and then stored at 4°C in a sterile tube. The viability of the cells was determined for a 2-week period of storage at 4°C. Figure 1 shows a summary of the titers of A. baumannii cells in suspension that were stored at 4°C for a >2-week period.

Coupon Preparation and Cell Inoculation

Hospital-relevant surfaces included painted and unpainted aluminum, unpainted steel, and scrub (laboratory coat material). Small pieces (2 × 1 cm), referred to henceforth as coupons, were cut, and metal coupons were precleaned with 70% ethanol and dried. Scrubs and precleaned metal coupons were sterilized through autoclaving in glass Petri plates.

The stock culture of A. baumannii was appropriately diluted to achieve two working stocks, with titers of 2 × 10^sup 6^ cells per mL and 2 × 10^sup 4^ cells per mL. Aliquots of 50 /µL of the two working stocks UVC Exposure

A special light exposure box, measuring 45 × 19 cm, was designed and fabricated by the Naval Surface Warfare Center (Dahlgren, Virginia). The ultraviolet lamp was hung with a beaded chain, and its vertical height from the floor could be readily adjusted. The floor area that received maximal irradiation with ultraviolet light spanned only 9 × 17 cm (13 cm from both sides of the box, 8 cm from the back side, and 2 cm from the front side). Figure 3 shows a schematic diagram of the floor and demarcation of the area receiving maximal fluence of ultraviolet light. The light bulb was positioned -4 cm above the floor. The numbers indicated in the inner rectangle are flux values (microwatts per square centimeter) at this height of the bulb. A sheet of corrugated packing material was placed on the floor, and the mapped area was cut out, to ensure placement of the coupons in precise locations every time. In preliminary studies, the flux and energy fluence (ultraviolet dose) were found to be constant for a period of 30 minutes at three different heights of the ultraviolet lamp (results not shown).