Ethanol and Isopropyl Alcohol Exposure Increases Biofilm Formation in Staphylococcus aureus and Staphylococcus epidermidis

Introduction Alcohols, including ethanol and isopropyl alcohol, are used in clinical practice for disinfection and infection prevention. Recent studies, however, demonstrate that alcohols may enhance biofilm production in Staphylococci. Methods We quantified biofilm formation in the presence of ethanol and isopropyl alcohol in six different, well-characterized strains of Staphylococcus epidermidis and Staphylococcus aureus. After 24 h of biofilm development, each strain was exposed to normal saline (NS), ethanol, or isopropyl alcohol (40%, 60%, 80% and 95%) for additional 24 h incubation. Adherent biofilms were stained and optical density was determined. Viability of strains was also determined after alcohol exposure. Results Ethanol increased biofilm formation in all six strains compared to normal saline (p < 0.05). There was increased biofilm formation with increasing ethanol concentration. Isopropyl alcohol also increased biofilm formation with increasing alcohol concentration in all six strains (p < 0.01 vs NS). The slime-negative, chemical mutant strain of S. epidermidis increased biofilm formation after exposure to both alcohols, likely reverting back its primary phenotype through modulation of the intercellular adhesin repressor. All strains demonstrated viability after exposure to each alcohol concentration, though viability was decreased. Conclusion Ethanol and isopropyl alcohol exposure increases biofilm formation of S. aureus and S. epidermidis at concentrations used in clinical settings. Ethanol and isopropyl alcohol did not eradicate viable Staphylococci from formed biofilm. Electronic supplementary material The online version of this article (doi:10.1007/s40121-015-0065-y) contains supplementary material, which is available to authorized users.


INTRODUCTION
Staphylococci, including Staphylococcus epidermidis and Staphylococcus aureus, are common biofilm-forming pathogens [1]. They frequently cause implant and catheterassociated infections, and are a significant cause of morbidity and mortality [1]. Previous studies have demonstrated increased biofilm production of S. epidermidis and S. aureus after exposure to different alcohols, including ethanol at concentrations above 40% [2,3]. This is important since isopropyl alcohol is commonly used as a cutaneous disinfectant and ethanol is used in catheter lock solutions for the treatment and prevention of catheter-related bloodstream infections (CRBSI) [1,4]. Although ethanol-based catheter lock solutions, including combinations with isopropyl alcohol, have been advocated for the prevention and management of CRBSI at concentrations between 25% and 100%, ethanol-based lock solutions may have unintended consequences since CRBSI are frequently caused by biofilmforming bacteria [5,6]. Additionally, ethanol use in lock solutions has been demonstrated to have other deleterious effects [5,6].
We compared the effects of ethanol and isopropyl alcohol on Staphylococcal biofilms using a semi-quantitative microtiter plate assay to better understand the effect of these alcohols on biofilm formation. We also measured the viability of biofilm-embedded bacteria after exposure to ethanol or isopropyl alcohol.

Medium
Strains were grown overnight on Tryptic Soy glucose, 2% sodium chloride, 25 mg/L calcium, and 12.5 mg/L magnesium was used to optimize biofilm production in the biofilm assay [12,13].

Viability
Viability of biofilm-embedded Staphylococci was evaluated using a similar 96 well plate assay [15]. After inoculation, incubation and alcohol or NS exposure as above, media was removed and wells were carefully rinsed three times with NS to remove planktonic bacteria. Wells were then filled with 200 lL of NS and plates were sonicated for 20 min in a water bath sonicator (Fisher Scientific FS20, Pittsburg, PA, USA) to disperse adherent biofilms. Viability was determined in quadruplicate on two occasions by plating aliquots from each strain and alcohol concentration. Plate counts were determined after 24 h incubation. The lower limit of detection for this method is 2.0 log 10 CFU/mL.

Statistical Analysis
OD and log CFU/mL were compared between groups using analysis of variance (ANOVA) with Tukey's post hoc test [16]. Data is presented as the mean OD with standard error of the mean using at least eight replicates for each strain and test solution combination. Statistical analysis was conducted using SPSS (release 20; SPSS, Inc. Chicago, IL). A p value of \0.05 was considered significant. Each alcohol concentration was compared to NS, and mean difference (change) in OD between alcohol and NS was determined, with a corresponding p value. Mean differences in OD are presented as a range for all the strains in the results.

Compliance with Ethics
This article does not contain any new studies with human or animal subjects performed by any of the authors.

RESULTS
Ethanol exposure increased biofilm in all strains ( Fig. 1a)

DISCUSSION
Our results are similar to a previous study demonstrating increased S. aureus biofilm formation after ethanol exposure [2], however, there are conflicting reports on the viability of those biofilm bacteria [17,18]. We found these bacteria within biofilm were viable, although viability was decreased compared to NS-exposed biofilm. In contrast to previous reports [4,19], bacteria in biofilm were not eradicated after alcohol exposure. This may be due to different methods used to remove the biofilm from 96 well plates, as prior studies removed biofilm using cotton swabs [4,19], whereas we sonicated the well plates. We also found an increase in biofilm formation with increasing alcohol concentration. Only one strain, the prolific biofilm-forming S. epidermidis, decreased biofilm formation with increasing concentrations of ethanol. This strain was likely near maximal biofilm production possible in this assay. Small variations in The bactericidal effect of alcohol depends upon dehydration and denaturation of proteins [20]. Mixtures of alcohols and water (60-90% v/v) are more effective because proteins are denatured more quickly in the presence of water [20,21]. Ethanol also causes leakage of the plasma membrane, disrupting bacterial growth and metabolism [22]. The impact of dehydration on cell death in the presence of alcohols may not be observed in catheter lock solutions since these do not dry, however denatured proteins and leaking membranes may still lead to decreased viability. The high concentrations of ethanol in catheter lock solutions increase biofilm formation in Staphylococci and also predisposes to catheter dysfunction and plasma protein precipitation [6].
Staphylococcus epidermidis M7, the isogenic  [23,24]. This strain does not accumulate on glass and polystyrene surfaces [23], but it accumulates on polyvinyl chloride disks and has been shown to produce biofilm [25][26][27][28]. The exact mechanism for the mutation is unknown but is believed to be due to alteration of the intercellular adhesin (ica) gene [10]. The ica gene regulates production of polysaccharide intercellular adhesin, the major exopolysaccharide produced in S. epidermidis and S. aureus biofilm [29]. Ethanol increases Staphylococcal biofilm formation by increasing ica expression through modulation of the repressor, icaR [2,3,29,30]. It is possible that alcohol exposure and subsequent increase in ica expression allowed accumulation and biofilm formation of this strain in polystyrene plates.
To our knowledge, this is the first report of any alcohol exposure to cause the M7 strain to increase biofilm formation. Regarding limitations, we tested a small number of strains, including one clinical isolate which may have different biofilmforming behavior. The crystal violet used in this study stains cells and does not differentiate between viable and nonviable cells or quantify extracellular matrix production. Also, we did not characterize the composition or matrix production of the biofilms. We considered that alcohol may denature bacteria in biofilm, allowing for greater penetration of the crystal violet. However, differences in biofilm formation could be observed between wells even before the crystal violet stain was added.
This also would not account for the increase in ica expression noted previously [30]. Viability may be underestimated using this method, since some adherent cells were visible in the bottom of wells after 20 min of sonication, particularly the prolific biofilm-forming ATCC 35984. Sonication of well plates can fail to release cells completely [31]. There was also a tendency for the number of bacteria to be higher in the center of the well plate than along the edges where evaporation was higher, further suggesting that dehydration played a role in cell viability.