Effect of alkaline pH on staphylococcal biofilm formation ANTONIA NOSTRO, 1 LUIGINA CELLINI, 2 MARA DI GIULIO, 2 MANUELA D’ARRIGO, 1 ANDREANA MARINO, 1 ANNA RITA BLANCO, 3 ANGELO FAVALORO, 4 GIUSEPPINA CUTRONEO 4 and GIUSEPPE BISIGNANO 1 1 Pharmaco-Biological Department, University of Messina, Messina; 2 Department of Drug Sciences, School of Pharmacy, University ‘G. d’Annunzio’, Chieti; 3 SIFI Pharma SpA, Catania; and 4 Department of Biomorphology and Biotechnologies, University of Messina, Messina, Italy Nostro A, Cellini L, Di Giulio M, D’Arrigo M, Marino A, Blanco AR, Favaloro A, Cutroneo G, Bisignano G. Effect of alkaline pH on staphylococcal biofilm formation. APMIS 2012; 120: 733–42. Biofilms are a serious problem, cause of severe inconvenience in the biomedical, food and industrial environment. Staphylococcus aureus and S. epidermidis are important pathogenic bacteria able to form thick and resistant biofilms on various surfaces. Therefore, strategies aimed at preventing or at least interfering with the initial adhesion and subsequent biofilm formation are a considerable achievement. The aim of this study was to evaluate the effect of alkaline pH on bacterial adhesion and further biofilm formation of S. aureus and S. epidermidis strains by biofilm biomass, cell-surface hydrophobicity, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) analysis. The results demonstrated that the amount of biofilm biomass formed and the sur- face hydrophobicity were significantly less than what were observed at higher levels of pH. SEM and CLSM images revealed a poorly structured and very thin biofilm (2.5–3 times thinner than that of the controls). The inhibiting effect of the alkaline pH on the bacterial attachment impaired the normal development of biofilm that arrested at the microcolony stage. Alkaline formulations could be promising towards the control of bacterial colonization and therefore the reduction of the biofilm-related hazard. In the clinical setting, alkaline solutions or cleaners could be promising to prevent the bacterial colonization, by treating surfaces such as catheters or indwelling medical devices, reducing the risk of biofilm related infections. Key words: Biofilm; pH; Staphylococcus aureus; Staphylococcus epidermidis; hydrophobicity; confocal laser scanning microscopy; scanning electron microscopy. Antonia Nostro, Pharmaco-Biological Department, Faculty of Pharmacy, University of Messina, Viale S.S. Annunziata, 98168 Messina, Italy. e-mail: anostro@unime.it Virulence of Staphylococcus aureus and S. epidermidis has been linked to the ability to form thick multilayered biofilms on different surfaces, cause of biofilm-associated infections and serious inconvenience in the environmen- tal, biomedical and industrial settings (1–5). Biofilm formation is a complex process that involves a sequence of events. It is the result of an initial attachment of the microrganisms to a surface, followed by an increase in the adherent population embedded in extracellular polymeric substances. Microbial adhesion to surfaces is influenced by many factors, inclu- ding physicochemical properties of both bacte- ria and substratum surfaces, bacterial ability to adhere to surfaces and also environmental factors (6). The primary attachment to sur- faces is a crucial event for the biofilm forma- tion. It is dependent on the non-specific interactions between bacteria and the surface, including van der Waals interactions, Received 10 November 2011. Accepted 20 February 2012 733 APMIS 120: 733–742 Ó 2012 The Authors APMIS Ó 2012 APMIS DOI 10.1111/j.1600-0463.2012.02900.x