Appl Biochem Biotechnol (2016) 178:1053–1067 DOI 10.1007/s12010-015-1928-0

Highly Bactericidal Polyurethane Effective Against Both Normal and Drug-Resistant Bacteria: Potential Use as an Air Filter Coating Matthew Taylor 1 & Bruce McCollister 2 & Daewon Park 1

Received: 7 August 2015 / Accepted: 9 November 2015 / Published online: 18 November 2015 # Springer Science+Business Media New York 2015

Abstract The battle against the prevalence of hospital-acquired infections has underscored the importance of identifying and maintaining the cleanliness of possible infection transmission sources in the patient’s environment. One of the most crucial lines of defense for mitigating the spread of pathogens in a healthcare facility is the removal of microorganisms from the environment by air filtration systems. After removing the pathogenic microorganisms, the filters used in these systems can serve as reservoirs for the pathogens and pose a risk for secondary infection. This threat, combined with the ever-growing prevalence of drug-resistant bacterial strains, substantiates the need for an effective bactericidal air filter. To this end, a broad-spectrum bactericidal polyurethane incorporating immobilized quaternary ammonium groups was developed for use as an air filter coating. In this study, the bactericidal activity of the polymer coating on high-efficiency particulate air (HEPA) filter samples was quantified against eight bacterial strains commonly responsible for nosocomial infection—including drug-resistant strains, and confirmed when applied as a filter coating in conditions mimicking those of its intended application. The coated HEPA filter samples exhibited high bactericidal activity against all eight strains, and the polyurethane was concluded to be an effective coating in rendering HEPA filters bactericidal. Keywords Pathogens . Antibacterial . Air filtration system . Drug-resistant strains . Coating

* Daewon Park [email protected] 1

Department of Bioengineering, University of Colorado Denver, Denver, CO 80206, USA

2

Microbiology Laboratory, University of Colorado Hospital, University of Colorado, Aurora, CO 80045, USA

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Introduction Nosocomial infection is a ubiquitous and immensely costly threat worldwide in terms of patient morbidity, mortality, and medical expenses [1–4]. Based on a survey conducted by the US Centers for Disease Control and Prevention (CDC) in 2011, healthcare-associated infection affects 4 % of all patients, totaling an estimated 648,000 patients with 721,800 healthcareassociated infections per year in the USA alone [5]. In 2007, the five most common hospitalacquired infections were estimated to account for $9.8 billion annually [4]. The vast majority of healthcare-associated infections are caused by bacteria, although viral and fungal infections can also be contracted in healthcare institutions [6, 7]. The continued therapeutic and prophylactic use of antibiotics to combat these infections has facilitated the emergence and increasing prevalence of drug-resistant strains of these common bacterial species. The medical equipment, air, floors, walls, and linens within the hospital are all potential sources of nosocomial infection as they are generally not sterile. The air in particular can transport infectious pathogens over both short distances by large droplets and longer distances by droplet nuclei generated by coughing or sneezing [8]. Droplet nuclei can also remain airborne for long periods and therefore disseminate over large areas [8]. Housekeeping procedures such as sweeping, mopping, and changing of soiled linen can also aerosolize particles containing pathogens which then may be directly inhaled or settle onto other surfaces [8, 9]. The institution must therefore be circulated with filtered air to dilute and remove airborne pathogens [8, 10–12]. Both the World Health Organization (WHO) and CDC state that in areas with increased susceptibility to infection such as hematology units, oncology units, and surgical wards, high-efficiency particulate air (HEPA) filtration must be used [8, 12]. HEPA filtration has been widely adapted in biomedical applications for the removal of infectious microorganisms from air circulated in healthcare institutions and microbiological laboratories [10–15]. According to standards set by the Department of Energy, HEPA filters must remove at least 99.97 % of airborne particles 0.3 μm in diameter. Since bacteria are on the order of 0.5–5 μm in size, HEPA filtration effectively removes bacterial pathogens from healthcare-associated environments’ air and prevents their release into downstream areas. After removing the bacteria from the air circulation, however, the HEPA filter essentially acts as a repository for the removed pathogens. Loaded with other biological waste removed from the air stream and often used in conjunction with humidifiers, the filters can facilitate not only the survival but also the multiplication of these pathogens [10, 11, 14, 15]. During routine filter maintenance or if the filter were to fail in some way, such as the formation of a tear in the media or the unseating of the media from the filter housing, there is a significant possibility of disseminating infectious bacterial pathogens into the air handling system that circulates throughout the institution. The serious threat and impact of nosocomial infection, combined with the ever-increasing prevalence of drug-resistant bacterial strains, only substantializes the necessity of—and risks involved with—maintaining HEPA filtration systems in healthcare settings. One approach to either minimize or eliminate this threat would be to create a filter media whose surface is lethal to bacteria—killing the bacteria after the filter captures them and preventing colonization. A disinfected filter would also be safe for both the worker and the healthcare facility during inspection and replacement of the filter. Coating the HEPA filter media with a bactericide would render the surface of the filter lethal to bacteria. Many systems have been developed as

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bactericidal coatings [16–30], although many existing coating systems are based on the incorporation of a bactericidal agent within the coating material and a subsequent release of the bactericide from the coating surface to exhibit bactericidal activity [19, 21, 24, 26–30]. Not only does this mechanism of action have a limited lifetime of activity as the bactericidal agent leaches away from the coating, it also releases potentially harmful bactericides (such as chlorine or bromine from N-halogen-based systems) into the environment. To address this shortcoming, we have created a non-leaching bactericidal polyurethane coating incorporating bactericidal quaternary ammonium groups within its polymer backbone. Polyurethanes are durable, simple to synthesize, and are known for their inherent durability [31], while, in general, polymer systems incorporating bactericides are ideally suited to such an application due to their inherent ease of customization in terms of the incorporated bactericidal agent and their degradation and release kinetics [22, 32]. Quaternary ammonium compounds, referred to as QACs, have shown high, long-lasting bactericidal activity and are non-toxic [22, 33–35]. Mechanistically, the electrostatic binding affinity of the quaternary ammonium cations with the negatively charged bacterial cell brings the cell outer envelope in contact with the polymer, forming interface complexes with the proteins and negatively charged lipid groups in the cell membrane that are enhanced by the penetration capability of the lipophilic alkyl chains. The negatively charged lipid groups then translocate from the inside leaflet to the outside leaflet and laterally segregate—compromising cell membrane structural organization and integrity, causing leakage of cytoplasmic constituents and cell lysis [22, 36–40]. The bactericidal quaternary ammonium groups are not leached away to exhibit bactericidal activity, and the coating therefore is effectively permanently bactericidal. To evaluate the developed polymer’s effectiveness as a bactericidal filter coating, the bactericidal activity of polymer-coated HEPA filter samples was observed against a multitude of bacterial pathogens commonly responsible for nosocomial infection—including Gramnegative and Gram-positive strains, clinical and standard strains, and drug-resistant strains. It was then verified that the bactericidal activity of the filter coating was retained when exposed to directional airflow mimicking the conditions of the filter’s intended application.

Materials and Method Materials Trifluoroacetic acid (TFA), agar powder, and iodomethane were obtained from Alfa Aesar (Ward Hill, MA). 1-Bromododecane and 4,4′-methylenebis(phenyl isocyanate) (MBPI) were obtained from Sigma-Aldrich (St. Louis, MO). Dimethylformamide (DMF) and tetrahydrofuran (THF) were obtained from BDH Chemicals (Poole, UK). Luria-Bertani (LB) broth was obtained from Bioexpress (Kaysville, UT). Phosphate-buffered saline (PBS, 1×, pH 7.4) was obtained from Corning (Manassas, VA). American Type Culture Collection (ATCC) supplied Escherichia coli (E. coli, Rosenbach strain, ATCC 15597); Staphylococcus aureus (S. aureus, ATCC 6538); S. aureus, Mu3 strain (resistant to methicillin, susceptible to vancomycin, ATCC 700698); and S. aureus, BAA-40 strain (resistant to methicillin, resistant to spectinomycin actinospectacin, ATCC BAA-40). Pseudomonas aeruginosa (P. aeruginosa, ATCC 27853), vancomycin-resistant Enterococcus faecalis (VRE, NJ-3 strain, ATCC 51299), Providencia stuartii (P. stuartii, ATCC 49809), and

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Serratia marcescens (S. marcescens, clinical isolate) were obtained from the Clinical Microbiology Laboratory at the University of Colorado Hospital. The air filter media used in the plate counting bactericidal activity assays and flow chamber tests were cut from a standard residential HEPA filter (model HRF-R1, Honeywell, Southborough, MA).

Synthesis of Q-PU The bactericidal polyurethane used in this study was synthesized as described previously [41]. Briefly, N-BOC serinol was reacted with MBPI in DMF to synthesize the base polyurethane. The BOC-protecting groups were then removed from the polyurethane by exposure to TFA. The regenerated primary amine groups were subsequently quaternized via successive alkylation with 1-bromododecane and iodomethane.

Q-PU-Coated Filter Preparation Filter samples were cut from a standard residential HEPA filter, immersed in a solution of the quaternized polyurethane (Q-PU) to fully saturate the filter fiber, removed, and air-dried until all solvent had been removed. The Q-PU was dissolved in a 7:1 mixture of THF/sterile water. Control condition filter samples were immersed in the THF/water solution containing no dissolved polyurethane and air-dried. In order to increase the thickness of the coating, coating solutions with 0, 2, 4, 6, 8, 10, 13, 25, and 50 mg/ml concentrations of polyurethane in the THF/water solution were used to coat filter samples.

Bactericidal Activity Assays Bacteria were proliferated from frozen monoclonal stock suspensions in Luria-Bertani (LB) broth (5 ml) for 16 h, shaking at 250 RPM and 37 °C. The bacterial suspension was centrifuged at 5000 RPM for 5 min, the LB broth poured off, and the bacteria resuspended in sterile PBS. The bacterial concentration of the PBS suspension was again estimated based on absorbance at a 600 nm and subsequently diluted to a 108 cells/ml suspension (the exact starting bacteria concentration was calculated after plate counting). Polyurethane-coated and uncoated filter samples were each immersed in 3 ml of bacterial suspension in 14-ml round-bottom tubes and incubated for 120 min, shaking at 250 RPM and 37 °C. At 0, 30, 60, and 120 min, 20-μL aliquots were removed from the bacterial suspensions and serially diluted in sterile 96-well round-bottom plates. Aliquots were diluted 10-fold at each iteration and dilutions were performed in triplicate. A 40-μl aliquot from each dilution was plated onto LB agar plates, allowed to dry, and incubated overnight at 37 °C. The colonies were counted from dilutions containing 20–80 colonies, and the concentration of viable bacteria in each bacterial solution was then calculated based on the number of colonies, the plated aliquot volume, and number of dilutions:

½Viable Cells ¼

ð# of ColoniesÞ*10# of Aliquot Volume

Dilutions

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Flow Chamber Tests A cylindrical testing apparatus was constructed using standard 4-in.-diameter polyvinyl chloride (PVC) piping sourced from a local hardware store (Fig. 1). One end of the chamber housed a fan obtained from a standard consumer hair drier that forced air into the cylindrical chamber through a diffuser to generate directional airflow. The opposite end of the chamber was closed except for outlets where HEPA filter samples could be mounted—forcing the directional air to pass through the filter samples. To evaluate whether the coated filter samples retained their bactericidal activity after exposure to directional airflow, a methicillin-sensitive S. aureus strain, Rosenbach, was proliferated from frozen monoclonal stock suspensions in LB broth for 16 h at 37 °C, shaking at 250 RPM. The bacterial suspension was then centrifuged at 5000 RPM for 5 min, the LB broth poured off, and the bacteria resuspended in sterile PBS. The bacterial concentration of the PBS suspension was estimated based on absorbance at a 600-nm wavelength (Synergy HT microplate reader, Biotek, Winooski, VT) and diluted to a 108 cells/ml suspension (again in PBS). Fifteen milliliters of this 108 cells/ml bacterial suspension was loaded into a small, finger-pump spray bottle and sprayed onto both surfaces of coated and control filter samples 5 cm in diameter. The bacteria-challenged filter samples were then exposed to airflow for 2 h in the flow chamber and removed from the apparatus. Rectangular sections 1×2.5 cm in size were cut from the center of each filter sample with a sterile scalpel and placed into 5 ml of LB in 14-ml round-bottom culture tubes and incubated overnight at 37 °C, shaking at 250 RPM. The presence of viable bacteria remaining on the filter samples was assayed by visually observing any turbidity of the LB after incubation. It was reasoned that if viable bacteria did indeed remain on the surface of the filter after flow chamber exposure, they would proliferate in the LB and be visually observable after overnight incubation.

Results and Discussion Determining Coating Thickness The ideal bactericidal air filter possesses both high antibacterial activity and high airflow efficiency. The bactericidal activity of the filter coating is dependent on physical interaction of Fig. 1 Cross-section view of flow chamber testing apparatus for mimicking typical airflow across HEPA filter samples

AIRFLOW( FAN

HEPA FILTER SAMPLES DIFFUSER

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bacteria with the surface of the polyurethane; therefore, the amount of available surface area of the coating correspondingly determines its bactericidal activity. Assuming the polymer coating on each filter fiber is roughly cylindrical, the coating surface area—and consequently the bactericidal activity—increases as the thickness of the coating increases at a factor of thickness2. Conversely though, as the coating thickness and filter fiber diameter increase, the pores become increasingly occluded (decreasing airflow efficiency) and the filter fiber structure is altered (decreasing filtration efficiency). Unlike typical membrane filters that remove particles in a sieve-like manner, HEPA filters remove particles from the air stream that are much smaller than both the size of the pores between the filter fibers and the diameter of the fibers themselves. When particles suspended in the air stream flow through the HEPA filter and contact the surface of the filter fiber, they are strongly held on the surface via Van der Waals forces and are effectively removed from the air stream [42]. Ultimately, the exact minimum filterable particle size depends largely on the fiber media structure and flow conditions—fiber diameter, filter construction, and flow velocity [42]. Assuming that the manufacturer of the HEPA filter material used in the study has already optimized the filter fiber diameter for maximum efficiency in removing suspended particles, as the media fiber structure is altered by the coating (i.e., increasing diameter due to coating thickness), the efficiency and performance of the coated HEPA filter media decreases. The optimal HEPA filter coating therefore must be sufficient enough in surface area so as to exhibit maximal bactericidal activity, however, must not be excessively substantial such that it impractically impedes the function of the filter media. To assess the bactericidal activity of the developed coating as a function of coating thickness, control and coated HEPA filters coated with varying concentrations of the quaternized polyurethane were immersed and incubated in S. aureus suspensions, and the concentration of viable cells was assayed over time (Fig. 2). Compared to uncoated control samples, filters coated with a coating concentration as little as 0.5 mg/ml of the polymer revealed a 10-fold reduction in viable bacteria by 30 min. The bactericidal activity of the coating increased as the concentration of the coating solution applied to the filter samples increased. The 4 mg/ml coating concentration was the most dilute concentration to kill all bacteria in solution—108 CFU/ml in 2 h, and all bacteria were killed within 30 min for coating concentrations of 8 mg/ml and higher. These results suggest that the increased bactericidal activity of higher coating concentrations was related to the increase in available surface area of the thicker coating. Compared to existing bactericidal surfaces, this represents very high bactericidal activity. The bactericidal activities of other antibacterial filters and surfaces range from less than 50 % decrease in viable bacterial cells to a 7-log reduction in viable bacteria from a 108 CFU/ml starting concentration [15, 20, 21, 24, 25, 43–47]. The structure of the coating at each thickness was imaged using SEM to observe the extent to which the structure of the filter fibers was altered (Fig. 3). SEM imaging confirmed that although higher coating concentrations resulted in increased bactericidal activity, the diameter of the fibers of the filter media progressively increased as coating concentration increased. Figure 3 highlights four of the eight coating concentrations imaged: 0 mg/ml (no coating control condition), 4 mg/ml, 8 mg/ml, and 13 mg/ml. The images gathered at these four coating concentrations show how the structure of the filter media fibers changed drastically as the coating thickness increased. Not only did the individual fibers increase in diameter, but they also began to agglomerate and web together. The beginning of this process can be seen in Fig. 3: in the images of the 8 mg/ml concentration coating, one can observe instances where two or three individual fibers have agglomerated together, whereas in the images of the

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Fig. 2 Bactericidal activity of quaternized MBP-PU-coated HEPA filter samples against S. aureus (non-resistant) at varying coating solution concentrations (i.e., varying coating thicknesses). Bactericidal activity is represented as the reduction in the concentration of viable cells in a 3-ml PBS suspension over a 2-h incubation with filter samples 1×2.5 cm in size. Bactericidal activity increases as the coating solution concentration was increased, with the uncoated control filter exhibiting no bactericidal activity

4 mg/ml concentration, this is not observed. The webbing effect as the coating concentration increases further can clearly be seen in the images of the 13 mg/ml coating concentration. These structural alterations of the HEPA filter associated with high polymer coating concentrations may drastically affect the performance of the filter media. Ultimately, a final coating concentration of 4 mg/ml was considered optimal as it exhibited rapid bactericidal activity and largely maintained the original structure of the filter media with minimal agglomeration or webbing.

Bactericidal Activity Assays The bactericidal activity of polymer-coated HEPA filter samples 1×2.5 cm in size was assessed against both methicillin-sensitive and methicillin-resistant strains of S. aureus. Compared to the 8-log reduction in bacterial concentration over 2 h observed against methicillin-sensitive S. aureus, the coated HEPA filters reduced the concentration of methicillin-resistant S. aureus strain BAA-40 by 4-logs (Fig. 4a). To further assess whether methicillin resistance in S. aureus may contribute to protection against the Q-PU, an additional bactericidal activity assay was performed against another methicillin-resistant S. aureus strain, Mu3. This strain was found to be as susceptible to polymer-mediated killing as that observed for the methicillin-sensitive S. aureus strain. These data suggest that the relative resistance observed for S. aureus strain BAA-40 appears to be independent of its resistance to methicillin. Since the bactericidal mechanism of the Q-PU targets and compromises the cell membrane via electrostatic and chemical interactions with the polymer’s quaternary ammonium groups [32, 40], variations in cell envelope structure may adversely affect the activity of the bactericidal coating. Gram-negative organisms contain both an inner cell membrane and outer membrane separated by a periplasmic space. This contrasts markedly with the singular cell membrane housed by Gram-positive organisms like S. aureus and E. faecalis. To assess the

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Fig. 3 SEM images of uncoated and coated HEPA filter samples at ×250, ×500, and ×1000 magnification. The thickness of the polymer coating was varied by altering the concentration of Q-PU in the solution used to coat the samples. Note the increasing degree of fiber agglomeration and webbing in addition to fiber diameter as the QPU concentration increased

susceptibility of Gram-negative organisms to the polymer coating, we evaluated the bactericidal activity of the coated filter samples against two common Gram-negative pathogens, E. coli and P. aeruginosa. Compared to the killing observed against the Gram-positive bacteria S. aureus, E. coli was less susceptible to the Q-PU, as the bacterial concentration of E. coli was reduced 4-logs over 2 h of incubation. When the assay was repeated using a larger-sized (3×2.5 cm instead of 1× 2.5 cm) coated filter, however, the entire E. coli population was eliminated within 30 min (Fig. 4b). Similarly, the larger-sized coated filter was highly bactericidal to P. aeruginosa, eliminating all viable bacteria following 60 min of incubation (Fig. 4b). Together, these data suggest that innate structural components of a bacterium might contribute to reduced susceptibility to the Q-PU, but in the case of Gram-negative E. coli, this appears to be overcome by using a larger surface area-to-bacteria ratio. Some alterations of bacterial membranes or cell wall structures are associated with resistance to various antibiotics. We sought to assess whether bacterial structural changes associated with antibiotic resistance might adversely affect the bactericidal

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Fig. 4 Concentration of viable bacterial cells in a 3-ml PBS suspension over a 2-h incubation with coated and control filter samples to evaluate bactericidal activity of quaternized Q-PU-coated HEPA filter samples against multiple bacterial pathogens commonly responsible for healthcare-associated infection, including drug-resistant strains. Possible effects on the antimicrobial activity of the filter coating conferred by methicillin resistance, possessing a Gram-negative cell wall, vancomycin resistance, and colistin resistance were analyzed in a, b, c, and d, respectively. For each data set, the shaded symbols refer to the bacterial concentration of uncoated, control filter samples and the non-shaded symbols refer to bacterial concentrations from coated filter samples

activity of our polymer. Vancomycin resistance, for example, is commonly observed in hospital strains of enterococcal species and is primarily mediated by alterations in the structural constituents of the peptidoglycan cell wall [48]. Despite altered cell wall structure, however, a vancomycin-resistant E. faecalis (VRE) strain, NJ-3, was highly susceptible to killing by the Q-PU coating on 1×2.5 cm filter samples, suggesting that alterations in peptidoglycan structure of vancomycin-resistant enterococcal isolates may not contribute to resistance to the Q-PU (Fig. 4c).

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ƒFig. 5

Investigation of possible causes of reduced susceptibility in colistin-resistant bacterial strains to the developed HEPA filter coating. a Reducing the starting bacterial concentration to 106 from 108 CFU/ml resulted in complete killing against both P. stuartii and S. marcescens by 60 min. b A bactericidal activity test performed on bacteria previously exposed to the filter coating, proliferated to a 108 CFU/ml concentration, resulted in no loss of susceptibility. These results suggest that the reduced susceptibility of colistin-resistant bacterial strains against the filter coating may be caused by a surface area-dependent reduction in the bacteria-coating interaction and that the filter coating does not facilitate the emergence of resistant bacterial populations. For each data set, the shaded symbols refer to the bacterial concentration of uncoated, control filter samples and the non-shaded symbols refer to bacterial concentrations from coated filter samples

Colistin is a polycationic antibiotic that is lethal to many Gram-negative organisms and has a similar mode of action as the developed Q-PU as it electrostatically targets the bacterial cell membrane and subsequently disrupts its integrity [49]. P. stuartii and S. marcescens are naturally multi-drug-resistant organisms, whose specific resistance mechanism to the polycation colistin has yet to be elucidated [50–52]. When incubated with the larger-sized 3×2.5 cm filter samples, the antimicrobial activity of coated HEPA filters was reduced against P. stuartii and S. marcescens as compared to that observed against intrinsically colistinsensitive E. coli and P. aeruginosa strains (Fig. 4d). Given their innate resistance to colistin, the significant numbers of surviving P. stuartii and S. marcescens bacteria observed following exposure to the polymer were not entirely unexpected. We wondered whether decreasing the bacteria-to-surface area ratio would result in improved bactericidal activity against these colistin-resistant bacterial strains, as was observed with the Gram-negative bacteria E. coli and P. aeruginosa. In this case, we choose to decrease the starting bacterial load, from 108 to 106 CFU/ml prior to exposure to the large coated HEPA filters. By decreasing the initial concentration of bacteria, both P. stuartii and S. marcescens were rapidly killed, with no viable bacterial cells remaining following 60 min of exposure to the polymer (Fig. 5a). Repeated bacterial exposure to antimicrobials can promote the emergence of resistance in these organisms. We wondered whether exposure to our developed coating might likewise facilitate the proliferation of resistant bacteria. In order to evaluate this possibility, we employed P. stuartii, the most innately resistant bacterial strain to the polymer in the study. A 108 CFU/ml population of P. stuartii was incubated with a polymer-coated HEPA filter sample until approximately 104 CFU/ml viable bacteria remained. A sample of the surviving bacterial cells were then proliferated for 16 h and exposed to the large coated HEPA filter at a starting concentration of 106 CFU/ml. All viable bacterial cells were eliminated within 60 min of exposure (Fig. 5b). These data suggest that repeated exposure to our Q-PU coating does not appear to select for increased bacterial resistance, even for a bacterial strain innately less susceptible to the polymer. Overall, our findings support the role of quaternized Q-PU as a bactericidal coating for HEPA filters with activity against common nosocomial Gram-positive and Gram-negative pathogens including those associated with antibiotic resistance. Ultimately, despite the fact that the filter coating appeared less effective against E. coli, P. aeruginosa, P. stuartii, and S. marcescens compared to methicillin-sensitive S. aureus, MRSA Mu3, and VRE, the magnitude of the bactericidal activities still represented high killing efficiencies. The lowest bactericidal activity encountered in this study, a roughly 2-log reduction against P. stuartii and 3-log reduction against S. marcescens, represents a substantial amount of killing considering a starting bacterial load of 108 CFU/ml. One study found that the bacterial load on the air filter media itself ranged between 103 and 105 CFU per gram and square centimeter [14]—loads

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which would also be easily eliminated by the developed filter coating. In general, all bacterial species and strains tested are pathogens very commonly responsible for nosocomial infections, with S. aureus, E. coli, E. faecalis, and P. aeruginosa being among the top six pathogens responsible for all healthcare-associated infections in the USA [5–7]. In addition, the increasing prevalence of and morbidity associated with drug-resistant bacterial strains cannot be understated. More than 80 % of strains of the previously susceptible species S. aureus are now resistant to benzyl penicillin [53], and from 1987 to 2003, the proportion of infections in intensive care units for MRSA has risen from roughly 20 to 40 % and from nearly 0 to 28 % for VRE [4].

Flow Chamber Tests The directional airflow apparatus (Fig. 6) exposed filter samples to conditions mimicking the conditions of the filter’s intended application. After compromising the HEPA filters with a suspension of methicillin-sensitive S. aureus and subjecting them to 2 h of constant airflow, sections of the filters were aseptically cut from the exposed filter samples and incubated overnight in LB broth. Following incubation, there was no observable turbidity in the medium that housed the coated HEPA filter. In stark contrast, there was gross turbidity seen in the LB broth that contained the uncoated control filter (Fig. 6). Similar results were obtained over a total of six experimental repetitions. These results reveal that the Q-PU retains its bactericidal activity as a HEPA filter coating during simulated airflow conditions.

Conclusions The developed quaternary ammonium-incorporating polyurethane coating both possessed bactericidal activity when tested in conditions mimicking its intended application as a HEPA filter coating, and exhibited very high bactericidal activity against a multitude of bacterial

Fig. 6 Appearance of LB broth from flow chamber tests after overnight incubation with the tested filter samples submerged. The coated filter sample is in the left tube and the uncoated control filter is in the right tube. Note the drastic contrast in turbidity of the broth between the two conditions. Viable bacterial cells remaining on the control filter after a 2-h exposure in the flow chamber were able to proliferate in the LB broth, whereas no noticeable growth in the LB containing the coated filter sample indicates the absence of viable bacteria on that filter’s surface

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strains commonly responsible for healthcare-associated infection—including Gram-negative and Gram-positive strains, clinical and standard strains, and drug-resistant strains. The magnitude of the bactericidal activity was high against each of the eight tested species/strains. This broad-spectrum activity, when examined in the context of the immense costs of nosocomial infection combined with the ever-increasing prevalence of drug-resistant bacterial strains, makes this Q-PU an ideal bactericidal filter coating for combatting the risk of secondary infection from contaminated filters. Acknowledgments This work was financially supported in part by State Bioscience Proof of Concept Grant (CU3460D) and University of Colorado Denver Start-up funding.

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