Healthcare Associated Infections (HAIs) are a major cause of morbidity and mortality in the United States [
1,
2]. HAIs were estimated to cause about 721,800 infections in acute care hospitals in 2011, and approximately 75,000 deaths each year [
3]. On average, one out of 31 hospital patients in the US contracts at least one HAI daily [
4]. Although it is estimated that 30–35% of HAIs can be prevented [
5], the occurrence of HAIs is still one of the leading causes of death in the US [
6].
Environmental surfaces (e.g., food trolleys, hospital commodes, bed rails) [
15] and mobile patient care equipment such as blood pressure cuffs [
16] and intravenous poles [
17] support the formation of biofilms by providing a platform for the attachment of planktonic bacterial cells [
9,
18,
19]. On any surface, biofilms are generally not evenly distributed [
20] and knowledge of the factors that influence the production of EPS by bacteria as
S. aureus under low shear conditions is limited [
21]. Once formed, biofilms are capable of growing on dry surfaces in healthcare facilities for protracted periods, surviving for as long as one year [
22,
23]. The definition of a DSB is controversial [
24,
25] as there is no clear definition or test method to differentiate varying degrees of dehydrated or dry biofilms. Dry surface biofilms (DSB) have been reported to persistently grow on contaminated environmental surfaces in healthcare facilities and can therefore play an important role in HAIs [
24]. Although dehydrated, DSB can still be characterized by the presence of EPS [
20,
26]. At maturity, bacterial cells in
P. aeruginosa [
27] and
S. aureus [
28] wet surface biofilms become motile, allowing for easy dispersal and re-attachment on other surfaces [
9,
13,
18]. More recently, a 2018 study by Chowdhury et al. demonstrated that DSB of
S. aureus can be dispersed to other surfaces even though they are dehydrated [
24]. However, the dispersal rates for DSB are relatively lower compared to the dispersal of planktonic bacterial cells [
24]. The use of antimicrobials to disinfect contaminated surfaces in healthcare facilities plays an important role in preventing HAIs [
13,
29,
30]. Studies have shown that wet surface bacterial biofilms can be more tolerant to disinfectants than planktonic cells [
31‐
33]. In an extensive review by Otter et al., mature wet surface biofilms were estimated to be on average 1000 times less susceptible to disinfectants than planktonic bacteria [
34]. The tolerance to antimicrobial disinfectants has been principally associated with EPS, which can shield underlying cells from direct contact with antimicrobials [
35,
36]. Compared to wet surface biofilms, dry surface bacterial biofilms can be more tolerant to disinfection procedures [
26,
37]. However, while there is an official method for testing the bactericidal efficacy of disinfectants against wet surface biofilms [
38], a comparable method for developing DSB for disinfectant efficacy testing in vitro has not been extensively developed. There are very limited studies that have developed a rapid in vitro DBS biofilm model and there is no clear standard for how many days or the conditions for a wet surface biofilm to dehydrate and become a DSB. Previous DSB development models include a dehydration step [
20]. Following ASTM E-2197 [
39], a two-hour drying time has been used to dehydrate wet surface biofilms for disinfectant efficacy testing [45]. A previously developed model required 12 days of repeated hydration and dehydration of bacterial cells to develop DSB [
20]. The objective of this study was to develop an in vitro model of
S. aureus and
P. aeruginosa DSB for disinfectant efficacy testing. In this study, we investigated the effect of different drying times and temperatures on the development of DSB of
S. aureus and
P. aeruginosa. We hypothesized that we would achieve a minimum six log
10 bacterial density on DSB per test coupon for disinfectant efficacy testing by subjecting wet surface biofilms to desiccation at near room temperatures. We also hypothesized that DSB of
S. aureus and
P. aeruginosa produced by the model in this study will be encased in EPS similar to those of wet surface biofilms to mimic associated disinfection challenges.