lu

D *ichig

al s

h care,inated

used decontamination techniques.12 Biolms in the hospitalenvironment not only increase patients likelihood of exposure of

disinfection, especially in the presence of high organicmatter loads.Thermal disinfection with steam autoclaves has been used fordecades to sterilize critical medical devices. The advent of mobile,portable steam generators has made steam disinfection of envi-ronmental surfaces much more practical.

A novel steam disinfection system developed by AdvancedVapor Technologies (Seattle, WA) equipped with proprietarythermal-accelerated nanocrystal sanitation (TANCS) technology

* Address correspondence to Chuanwu Xi, PhD, 6626 SPH Tower, 1415Washington Heights, Ann Arbor, MI 48109-2029.

E-mail address: cxi@umich.edu (C. Xi).Partially supported by a grant from Advanced Vapor Technologies, which

provided the steam vapor device used in the study but was not involved in the

Contents lists availab

American Journal of

journal homepage: w

American Journal of Infection Control 40 (2012) 926-30study design, data analysis, or data presentation.Contaminated surfaces are commonly linked to disease trans-mission,1-4 and environmental cleaning is important for controllinghospital-acquired infections.5-7 Bacteria grow in the hospitalenvironment mainly attached to surfaces as biolms,8 aggregates ofactive cells embedded within a polymeric matrix and attached toa biotic or abiotic surface. Biolms can act as reservoirs of patho-gens in the hospital environment and offer favorable environmentsto enable pathogens to persist for extended periods. The biolmstructure also protects embedded pathogens against manyantimicrobial agents, such as antibiotics and biocides,9-11 makingit very difcult to eradicate these pathogens with commonly

responsible for approximately 65% of nosocomial infections ; thus,developing effective procedures to combat biolms in the hospitalenvironment to control hospital-acquired infections is of criticalimportance.

Liquid chemical disinfectants, such as sodium hypochlorite,quaternary ammoniums,14 phenolic disinfectants,15 hydrogen per-oxide, and silver ions,2 are usually used to decontaminate envi-ronmental surfaces, and the use of these compounds is periodicallyreviewed by the Hospital Infection Control Practices AdvisoryCommittee.16,17 These compounds are often toxic to humans andinefcient in eradicating biolms, or require long contact times forservice settings are often contam

Environmental surfaces in healtConict of interest: None to report.

0196-6553/$36.00 - Copyright 2012 by the Associadoi:10.1016/j.ajic.2011.11.013equipped steam vapor technology against biolms through disinfecting biolms developed by 4 bacterialstrainsdEscherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, and Staphylococcusaureusdon an identical test surface (ie, polycarbonate) and biolms developed by E coli on 4 differenttest surfaces: polycarbonate, rubber, stainless steel, and ceramics.Results: Our data show that a 3-second steam treatment rapidly killed each biolm tested (>99.95 %killing efciency). For biolms developed on different surfaces, 3-second steam treatment achieved99.95% killing of E coli biolms developed on different surfaces. Compared with chemical disinfection,steam treatment for

and bleach (against E coli biolm). E coli biolms developed onceramic, stainless steel, rubber, and polycarbonate coupons wereused to evaluate the disinfection efcacy of the TANCS-equippedsteam vapor device against biolms developed by the same strainon different surfaces. E coli was chosen for this test because it isoften used as an indicator strain for water and environmentalcontaminations.

Disinfection

The coupons with well-developed biolms were placed ina 60 15mm sterile polystyrene dish and disinfected on both sideswith the steam vapor device. After 1 second, 3 seconds, and 5seconds of steam vapor disinfection, the treated coupons weretransferred into a Corning tube containing 2 mL of 1 PBS buffer.The biolm biomass was released from the coupons by vortexingfor 10 seconds, followed by homogenization. The homogenizedmixtures were serially diluted and plated onto LB agar plates.Viable cells were determined by counting colonies formed on theagar plates. The disinfection efciency was expressed as log

f Intypes of microorganisms, reducing the risk of surface-mediatedinfections.18 The TANCS technology uses the naturally occurringminerals in tap water to form crystals. As these crystals passthrough the boiler, they gain energy from the heat. When the watertransforms into superheated low-moisture steam (dry steam),these energized crystals are accelerated along with the steam.19

This process helps disrupt the cell membrane, allowing lethaltemperatures to quickly destroy bacterial cells. This novel steamvapor disinfection technology is considered to have great potentialfor eliminating biolms on environmental surfaces in the afore-mentioned settings; however, a detailed study on the efcacy ofthis device against biolms has not yet been reported.

Escherichia coli, Acinetobacter baumannii, Staphylococcus aureus,and Pseudomonas aeruginosa are important epidemiologic patho-gens commonly detected on environmental surfaces.20-23 Wetested the disinfection efcacy of the TANCS technology against thebiolms developed by these bacterial strains. Given that varioussurfaces inuence the initial adhesion and colonization of bacteriaand consequently affect biolm development and structure,24 wealso evaluated the disinfection efciency against biolms devel-oped by E coli on 4 different test surfaces commonly found in thehospital environment.

MATERIALS AND METHODS

Bacterial strains

Four bacterial strains were tested in this study. E coli K-12MG16653, A baumannii (ATCC 17978), and S aureus (ATCC 45330)were purchased from American Type Culture Collection (Mana-ssas, VA). P aeruginosa PAO300, a mucA22 derivative of P aerugi-nosa PAO1 that constitutively overproduces alginate,25 wasobtained from the University of Copenhagen. Luria Bertani (LB)medium (broth and agar) was obtained from Fisher Scientic(Pittsburgh, PA).

Chemical disinfectant

Bleach (5% sodium hypochlorite) was purchased from FisherScientic. Chemical disinfection was performed according to themanufacturers instructions.

Biolm reactor

A 1-L Centers for Disease Control and Prevention (CDC) biolmreactor with a coupon holder and 10-mm-diameter coupons ofdifferent materials (polycarbonate, rubber, stainless steel, andceramic) were purchased from BioSurface Technologies (Bozeman,MT). The CDC biolm reactor was developed to provide consistentbiolm samples and growth conditions for evaluation of antimi-crobial agents, surface treatments, and materials as described inASTM 2562-07, Standard Test Method for Quantication of Pseu-domonas aeruginosa Grown with High Shear and Continuous FlowUsing a CDC Biolm Reactor.

Steam vapor device

The steam vapor device (MondoVap 2400 commercial modelequipped with TANCS component SV-C/T) was provided byAdvanced Vapor Technologies (Seattle, WA). For the tests describedherein, the unit was outtted with a hose connected to a 14 14 14 cm triangular cleaning head. A cotton terry towel that had been

L. Song et al. / American Journal omachine-washed with ordinary detergent was afxed to thecleaning head in accordance with the manufacturers instructions.Before the experiments, the unit was lled with ordinary tap water,activated, and allowed to reach the functional operating boilerpressure of 66 psi. The steam delivery output was set to 12-15 psifor all experiments.

Biolm development

All biolms for the study were developed in the CDC biolmreactor. For this, 10% LB medium was pumped into the CDC reactorcontinuously at a ow rate of 120 mL/h using a mini pump (FisherScientic). The reactor has a residual volume of 400 mL anda hydraulic retention time of 3 hours, 20 minutes. The stirring ratewas maintained at 50 rpm for all experiments. Test bacteria weregrown in LB medium at 37 C overnight, after which 4 mL of theovernight culture was injected into the CDC reactor for inoculation.Biolms developed over 4 days after inoculation for E coli and over5 days after inoculation for A baumannii, S aureus, and P aeruginosawere used for the disinfection experiments.

Biolms composed of the individual test organisms of E coli,A baumannii, S aureus, and P aeruginosa grown on polycarbonatecoupons were used to evaluate the disinfection efcacy of theTANCS-equipped steam vapor device (against different biolms)

Table 1Disinfection efcacy (expressed as log reduction, with coefcient of variation inparentheses) of dry steam against different biolms developed on the polycarbonatesurface

Biolm Treatment time, seconds Disinfection efcacy

E coli 0 0.0 (0.49)1 3.8 (0.27)3 5.0 (1.41)

10 6.87P aeruginosa 300 0 0.0 (0.12)

1 3.0 (0.20)3 4.2 (0.14)5 5.3 (0.38)

S aureus 0 0.0 (0.29)1 2.9 (0.39)3 4.7 (0.26)5 5.3 (0.43)

A baumannii 0 0.0 (0.18)1 2.6 (0.38)3 4.1 (0.17)5 4.9 (0.27)

fection Control 40 (2012) 926-30 927reduction (log10 N/N0, where N is the number of viable cells afterdisinfection and N0 is the number of viable cells in biolms oncoupons used as controls without disinfection).

lm

f InFor chemical disinfection of biolms, coupons with well-developed biolms were immersed in 2 mL of chemical disinfec-tant solution (10 ppm sodium hypochlorite) and incubated for 10

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Fig 1. Disinfection efciency (expressed as log reduction) of steam against different bioSa, S aureus.

L. Song et al. / American Journal o928minutes and 20 minutes at room temperature. After treatment, thecoupons were gently washed 3 times with 1 PBS buffer to removedisinfectant residuals, and then transferred to a Corning tubecontaining 2 mL of 1 PBS buffer. The viable cells in biolms anddisinfection efciency were determined as described earlier.

All disinfection experiments were done in duplicate, andduplicate agar plates were used to count viable cells for eachdilution. The coefcient of variation (CV) was used to evaluate thereproducibility of viable cell counts (Tables 1).

Microscopic examination of biolms

To visually evaluate the efcacy of steam vapor disinfection,E coli biolms developed on different coupons without disinfectiontreatment (controls) or after 3-second disinfection treatment withthe steam vapor device were stained in the dark for 20 minuteswith LIVE/DEAD BacLight bacterial viability dye (Invitrogen,Carlsbad, CA) in accordance with the manufacturers instruction.The coupons were examined with a confocal scanning microscope(Olympus FV500/IX; Tokyo, Japan) equipped with detectors andlter sets for monitoring SYTO-9 and propidium iodide. Imageswere obtained using a 60 objective only from areas where biolmcells were readily detected. Simulated 3D images were recon-structed using the Amira software package (Amira, San Diego, CA).

RESULTS

Disinfection efcacy of dry steam against biolms developed by 4different bacterial strains on polycarbonate coupons

To test the disinfection efcacy of dry steam against biolmsdeveloped on the polycarbonate surface, biolms developed by the4 test strains on polycarbonate coupons were disinfected with thesteam vapor device for 1-10 seconds. Without disinfection treat-ment, different strains yielded different biolm biomasses on the

0 1 3 5-6

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s developed on the polycarbonate surface. PAO 300, P aeruginosa 300; Ab, A baumannii;

fection Control 40 (2012) 926-30polycarbonate coupon (E coli, 7.4 106 CFU/coupon; A baumannii,3.6 107 CFU/coupon; S aureus, 1.6 108 CFU/coupon; andP aeruginosa, 4.3 107 CFU/coupon, which were set as N0 for thecalculation of log reduction). All test biolms were killed rapidlythrough very brief applications of steam vapor generated from thedevice (Fig 1 and Table 1). A >4 log reduction (99.99% killing) wasachieved with a 5-second steam vapor treatment (Table 1).Complete killing of E coli biolms to nondetectable levels wasachieved with a 10-second steam vapor treatment (Table 1). Anaverage 5.2 log reduction (99.999% killing) was achieved witha 5-second treatment for each of the 4 tested biolms (Table 1).

Comparing disinfection efcacy against biolms by chemicaldisinfectant and dry steam

Disinfection of biolms formed by E coli on the polycarbonatecoupon by bleach was also tested. A 1.8 log reduction (w99%killing) of E coli biolms was achieved using 10-minute 10-ppmsodium hypochlorite treatment. A longer incubation time (20minutes) did not improve disinfection efcacy (Fig 2A). Incomparison, only 1 second of steamvapor disinfectionwas requiredto reach a similar level of disinfection of E coli biolm (Fig 2B andTable 1).

Disinfection efciency of dry steam against E coli biolms developedon different surfaces

E coli formed biolms with different structures on each of the 4different test surfaces (rubber, ceramic, stainless steel, and poly-carbonate coupons; Fig 4) and yielded differing amounts of biomass(2.6 107 CFU/coupon on rubber, 9.9 107 CFU/coupon on ceramic,1.7 107 CFU/coupon on stainless steel, and 4.1 107 CFU/coupon

-6

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Treat Time (mins)Fig 2. Comparison of disinfection efciency (expressed as log reduction) of chemical dis

Stainless SteelCeramics Rubber Polycarbonate

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L. Song et al. / American Journal oon polycarbonate coupons, which were set as N0 for the calculationof log reduction). A >3 log reduction (w99.95 %) of E coli biolmswas achieved with the 3-second dry steam treatment (Fig 3). Interms of the impact of surface type on biolm killing efciency, thebest efcacy was observed against biolms developed on theceramic surface, followed by those on the stainless steel, poly-carbonate, and rubber surfaces (Fig 3). The 3D images of biolmsobtained before and after disinfection treatment indicate thatalmost all of the biolm cells remained were killed with the3-second dry steam treatment (Fig 4). Approximately 80% of thetreated surface was free of biolm cells, suggesting that a 3-secondsteam treatment can remove themajority of attached cells from thesurfaces in addition to killing these cells.

DISCUSSION

Hospital-acquired infections are a signicant concern in todayshealth care environment. Biolms are the primary habitat for manymicroorganisms. Biolms can protect microbes, including humanpathogens, in the environment from the actions of antibiotics andbiocides and may even release a more infectious dose back into theenvironment. Effective disinfection of biolms poses a challenge.Chemicals are widely used for disinfection of environmentalsurfaces, but their killing efcacy, especially against biolms, isquestionable. Moreover, residual chemicals pose a health risk,26,27

and certain chemicals have the potential to create cross-resistance

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Fig 3. Disinfection efciency (expressed as log reduction) of steam with a 3-secondtreatment against E coli biolms developed on different surfaces.to antibiotics.20,28 Steam disinfection does not carry such risks,using no chemicals, only water.

Bacteria embedded in biolms are more resistant to disinfec-tants compared with planktonic cells in the same biolms.29,30 Inone study, disinfection with bleach achieved a 5 log reduction of

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ctant (A) and steam (B) against E coli biolms developed on the polycarbonate surface.

Fig 4. Confocal scanning micrographs of E coli biolms developed on various surfaceswithout (upper row) and with 3-second dry steam treatment (lower row). Green dotsindicate live cells, and red dots indicate dead cells.

fection Control 40 (2012) 926-30 929E coli O157:H7, S aureus, and P aeruginosa suspended cells witha 30-second treatment3; however, in another study, bleach wasable to achieve only a 2 log reduction of bacterial cells (developedby Listeria monocytogenes, Pseudomonas fragi, and Staphylococcusxylosus) in biolm.31 A similar trend was observed with steamvapor disinfection. A 5-second treatment was needed to achieve100% killing of E coli suspended cells using the same steam vapordevice,18 whereas a 10-second treatment was required to achieve100% killing of E coli biolms.

Sufcient chemical disinfection usually requires a long reactiontime. Silver disinfectant technology has emerged as an effectivemethod for environmental surface disinfection, but one studyfound that 30 minutes were required to achieve a 2.2 log reductionof P aeruginosa and a 3.3 log reduction of S aureus.2 In the presentstudy, dry steam disinfection had robust disinfection efcacy, withonly a 3-second steam treatment needed to kill >99.95% of bacte-rial cells in biolms developed by the different bacterial strains, inagreement with the previous work on disinfection of suspendedbacteria.18 Bleach is the most widely used chlorine-based disin-fectant. Comparing steam vapor disinfection and bleach disinfec-tion, a

disinfecting surfaces of large areas composed of different materialsin the hospital environment.

E coli, methicillin-resistant S aureus, A baumannii, and P aeruginosaare all linked to nosocomial infections.32-34 Proper environmentalcleaning is critical to controlling hospital-acquired infections ofthese pathogens.5,6 Our ndings demonstrate that the dry steamvapor treatment can eliminate biolms formed by these bacterialstrains on various surfaces, suggesting that dry steam disinfectionhas great potential for controlling hospital-acquired infections. Allour tests were performed in a laboratory system, and further testsin clinical settings would strengthen the potential application of

11. OToole G, Kaplan HB, Kolter R. Biolm formation as microbial development.Ann Rev Microbiol 2000;54:49-79.

12. Vickery K, Ngo Q-D, Zou J, Cossart YE. The effect of multiple cycles ofcontamination, detergent washing, and disinfection on the development ofbiolm in endoscope tubing. Am J Infect Control 2009;37:470-5.

13. Potera C. Forging a link between biolms and disease. Science 1999;283:1837-9.

14. Shirai J, Kanno T, Tsuchiya Y, Mitsubayashi S, Seki R. Effects of chlorine, iodine,and quaternary ammonium compound disinfectants on several exotic diseaseviruses. J Vet Med Sci 2000;62:85-92.

15. Gulati BR, Allwood PB, Hedberg CW, Goyal SM. Efcacy of commonly useddisinfectants for the inactivation of calicivirus on strawberry, lettuce, anda food-contact surface. J Food Prot 2001;64:1430-4.

16. Centers for Disease Control and Prevention. CRoCatHICPAC. Guidelines for

L. Song et al. / American Journal of Infection Control 40 (2012) 926-30930this new technology.

CONCLUSION

Our data show that the dry steam rapidly (with 3-secondtreatment) and effectively (>99.95% killing) killed E coli, Abaumannii, S aureus, and P aeruginosa biolms formed on thepolycarbonate surface and E coli biolms developed on the rubber,ceramic, stainless steel, and polycarbonate surfaces. Steam treat-ment for