LANGMUIR aubsacergicangnur Influence of Surface Roughness, Nanostructure, and Wetting on Bacterial Adhesion Minchen Mu," Shuhao Liv," William DeFlorio, Li Hao, Xunhao Wang, Karla Solis Salazar, ‘Matthew Taylor, Alejandro Castillo, Luis Cisneros-Zevallos, Jun Kyun Oh," Younjin Min,* and Mustafa Akbulut* cx This: hetps//dolorg/101021/aeslangmi 300091 O Read Online ACCESS| ABSTRACT: Bacterial fouling is a persistent problem causing the deterioration and failure of functional surfaces for industrial equipment/ components; numerous human, animal, and plant infections/diseases; and ‘encegy waste du to the ineficiencies a internal and external geometries of transport systems. This work gains new insights into the effect of surface roughness on bacterial fouling by systematically studying bacterial adhesion ‘on model hydrophobic (methy-terminated) surfaces with roughness scales spanning ftom ~2 nm to ~390 nm. Additionally, a surface energy integration ffamework is developed to elucidate the role of surface roughness on the energetics of bacteria and substrate interactions. For a sven bacteria type and surface chemistry; the extent of bacterial fouling was found to demonstrate up to a 7S:fold variation with surface roughness. For the eases showing hydrophobic weting behavior, both increated effective surface area with increasing roughness and decreased activation energy with increased surface roughness was concluded to enhance the extent of bacterial adhesion, For the cases of supeshydrophobic surfaces, the combination of factors including () the surpasing ‘of Laplace pressure force of interstitial air over bacterial adhesive force, (i) the reduced effective substrate area for bacteria wall due to air gaps to have drect/soli contact, and (i) the reduction of attractive van dee Waals force that holds adhering bacteria on the substrate were summarized to weaken the bacterial adhesion. Overall this study is significant inthe context of designing antifouling coatings and systems as well as explaining variations in bacterial contamination and biofilm formation processes on functional surfaces Li Metrics & More | GHaricte Recommendations — | @ Supporting mnformation ir as @ INTRODUCTION For abiotic surfaces, bacterial adhesion (attachment) is a major problem leading to pathogenic contamination and/or perform- ance losses of various devices, equipment, components, accessories, and vehicles used in healthcare, food and food packing, pipelines, and the maritime transportation indus tries." For biotic surfaces, bacterial adhesion is the fist step in bacterial infections, vegetable and fait spoilage, and plant Aliseases:*~* At a colloidal level, bacterial adhesion to a surface corresponds to a transition from the planktonic state to the sessile state, involving several phases: (i) diffusive, convective, ‘or flagellar motion of bacteria feom bulk fluid until bacteria start to sense nanoscale intermolecular forces between bacteria and the surface, (W suriace due to the gradient of intermolecular interactions between bacteria and the surface along with the thermal ‘energy, aT, and flagellar kinetic energy (If present), (ii) approach of the bacterial cell wall or its appendages to the surface until the disjoining pressure of displacing the surface hydration layer is overcome, and (iv) molecular contact and ¢ ACS Publications ™Aretercenalsicay translocation of bacteria toward the anchoring between the substrate and bacterial wall/appen ages Prior studies have reported that there are several key properties and parameters of bacteria influencing bacterial auhesion toa surface, each to varying degres. These include the hydrophobicity of the bacterial wall” surface potential of the bacteri"” bacterial size,"~"* bacterial shape,"'-"* the presence of curl and pili —"* quorum sensing ability” and the ability to produce extracellular bacterial adhesins."” In action, the characteristics of the substrate surface such as hydrophobicity," surface potential and charge,* hetero geneity, "* patterns" oughness,!°—"" and stillness" have been demonstrated to also be important. Some of the key extemal conditions governing bacterial adhesion trends are Received: Jamoary 10, 2023 Revised: March 22, 2023Langmuir suspension pH,"°* ionic strength," temperature,” and the characteristics of any convective flow fields present. ‘While there exist a large body of literature focusing on the influence of surface roughness on bacterial adhesion, the findings are conflicting, For instance, Yoda etal.” investigated the effect of roughness on adhesion of Staphylococcus «epidermidis to oxidized zirconium—niobium alley (arithmetic mean suriace roughness, or Ra, of 8.5 and 30.0 nm), cobalt chromium=molybdenum alloy (Ra of $8 and 12.0 nm), titanium alloy (Ra of 7.1 and 16 nm), pure titanium (Ra of 5.6 and 22.0 nm), and stainless steel susfaes (Ra of 1.8 and 7.2 mm). They found that there was increased bacterial adhesion on coarse surfaces compared to fine surfaces. Bohine ct al” prepared glass surfaces with five diferent roughnesses (0.07 yim, OS8 jm, 099 pm, 28 pm, and 5.8 ym) and observed that the rate of Escherichia coli (E.coli), Staphylococcus aureus (S. aureus), and Pseudomonas aeruginosa (P. aruginess) adhesion increased with increasing surface roughness. How. ever, Scheuerman eta relied on silicon surfaces with 10, 20, 30, and 40 jum wide grooves of 10 ym depth (ic, dlifering spacing parameters) to study the bacterial attachment bebavior of P. aeruginosa and Pseudomonas fluorescens. They found that the rate of bacterial attachment was independent of groove size for all bacteria, Hilbert et al” reported that the adhesion of Pseudomonas sp, Listeria monocytogenes, and Candida lipaytica to stainless steel surfaces was not influenced by surface roughness in the Ra range of 0.01 to 0.9 jm. On the other hhand, Truong etal.” compared the adhesion of P. aeruginosa and S. aureus on titanium surfaces with root-mean-square (RMS) roughness of 23.3 and 84.2 ne. They observed that S aureus and P. aeruginosa demonstrated preferential attachment to the smoother titanium surfaces that were prepared by cqual channel angular pressing. We etal.” reported that the number of adherent P. aeruginosa and S. aureus was significantly lower con rough stainless-steel surfaces as compared to the electro: polished, smooth stanless-stel surfaces. Encinas et a” have recently reported that bacteria adhesion can be suppressed by submicrometerscale surface roughness. Similarly, Jang et al.” have incorporated nanotexture on stainless stel surfaces via electrochemical etching to inhibit bacterial adhesion, To assess such paradoxical seeming trends of bacterial adhesion with cespect to surface roughness observed in the lnerature, we have relied on hydrophobized quartz surfaces with uniformly controlled surface chemistry and coverage but systematically varying surface roughness. Fourteen different surface roughness values to cover oughness at multiple length scales were utilized for this investigation. The methyl terminated substrates were particulasly selected due to their inert nature and since specific ligand-receptor interactions between such substrates and bacteria do not emerge. Hence, these surfaces allow us to clearly elucidate the inluence of surface roughness on bacterial adhesion to abiotic surfaces that is mostly controlled by nonspecific interactions. As bacterial microorganisms, Gram-negative Salmonella typhimurium LT2 (Salmonella) and Escherichia coli O1S7:H7 (B. colt) as well as Gram-positive Listeria innocua (Listeria) have been utilized With these selections, it is ensured that all bacteria bave a similar shape (ie, bacillus shape) and the effect of shape is not aalditionally superimposed to the influence of surface rough ness, In addition, Salmonella, E.coli, and Listeria are three key rierobial pathogens that are commonly associated with foodborne illnesses" While the characterization of surface chemistry and coverage was carried out using a scanning Xray pubs acs. org/Langmuir photoelectron spectroscopy microprobe (XPS), surface rough: nest was determined using atomic force mieroscopy (AEM). Dacterial adhesion was quantiied using direct visualization via scanning eleetron microscopy (SEM) Ml EXPERIMENTAL METHODS: Materials. Quarts (S10) was puchated (rom Ted Pell, ne (Reding, CA, USA). Tetrafnoromethane (CF) and oygen (0) fa war obtained fom Bras Valley Welding Supply ne and weed ‘irc s purchased (Bryan, College Staton, TX, USA). Possum iydronde (98%, reagent gra) war purchased fom War's Scenee (Rochester, NY, USA), Tretia clorde (TMCS, 2 950%) was obained fom Sigma-Aldrich Co. (St Loui, MO, USA) and wed vith the hexane (29508) purchased fom Avast Perormance Matera, Ine (CenterValey, PA, USA). The 200 proof pure ethanl (HPLC grade) wtlved war ordered from Koptee (King of Prosi, PA USA). For bacterial experiments, type soy agae CISA), type soy broth (TSB), and TSB containing O6% yeast exact (TSB-E) wrete procured fom Becton, Dickinson and Co, (Spck, MD, USA) Preparation of Quartz Surfaces with Different Surface Roughness. Quart sides were fist red wi lteapure water, (cesiety 2182 Mftem) called fom a water parction sytem (Sal. Advantage AIO; EMD Millipore Corp, Bilesica, MA, USA) ated then died tfoom temperatee, Te died slider weve subjected to orygen plums treatment conducted In order to remove organic deorbate ‘sng the CS-I7O1 reacivedon etther (RIE, Noadson MARCH, Concord, CA, USA) Following plasma ueatnent the quate slides were snsed with Mil Q water again and dried. Plasma frentment sever a dual parpes: not only ineentr the resciey of surface goups but ao inactivates any pre-easing bacteria on the faces" To produce suraces eth varying nanoroughesy, the prepared quarts des were tested inthe C8170 reactveion echt, nde CP. /0 gas with varying etching times sp to 2b Hydrophobization of Quartz Surfaces. To incease the hydeophobicty of the sacs, the roughened. quarts sider were sod sing TMCS, which was prepared by disting TMCS (6 we Sin Beane The quater slides were then dipped into the TMCS and hexane solution for 24 h to alow the alsnation modieston of the ‘faces to occ, Next the samples were removed fromm the TMCS folation and rinsed three tires with ethane] to remove excess TMCS tnd byprodvets Atl, the samples were dned with comprested Aluogen gar before characesiation, ‘Characterization of Surface Roughness. The morphology and seughoee of quart cer with dfventreuphonene were fied Suing stomie force microscopy (APM, Braker Dinension leon Bilericy MA, USA) in tapping mode, Severl parameters were sqsnted to analyse the sce roughness, ach a the rot ena ‘are (RMS) roughness, astocodation length, and roughness at The RMS roughness war clelated based on the root mes sqeae oF the height of microscale peaks and wales as means of guantving the average feature ste, The roughness ttl wat ealeslated by Alviding the actoal sure area tothe projeced aren (@ 2 1). [Astocorraton length wae obtained fom the anaie of power spectal density function (PSDT). The Gwyddon software 249 (Caech Metrology Institute, Java, Cech Republi) war sed to analyse the AFM imager and calelate the abovementioned Parameter vals foreach spl. ‘Characterization of Surface Chemistry, The chemical iter actions between the modiied quarts and TMCS were studied va derwated ttl rellectnce Rourer taf ined spectroscopy (ATRETIR) sung the IAPretige- 21” (Shimaday Corp, Kyoto, Japan) systema The resus of ATRCFTIR were analaed with the ‘Reolaon version LAD (Shimadsu Corp, Kyoto, Japan) software TMCS coverage onthe modied surfaces a charcienaed lng PLM VesaProe Il scaming XPS microprobe (Physical Hecronis Chanhassen, MN, USA) The ATR-PTIR and XPS rests ate show the Supporting Information to indies the success modietion the voiene a wll asthe nr chr propane tnd covemg> of the surce (igure SI and Figure 52)