Biofilms are widespread in nature and constitute an important strategy implemented by microorganisms to survive in sometimes harsh environmental conditions. They can be beneficial or have a negative impact particularly when formed in industrial settings or on medical devices. As such, research into the formation and elimination of biofilms is important for many disciplines. Several new methodologies have been recently developed for, or adapted to, biofilm studies that have contributed to deeper knowledge on biofilm physiology, structure and composition. In this review, traditional and cutting-edge methods to study biofilm biomass, viability, structure, composition and physiology are addressed. Moreover, as there is a lack of consensus among the diversity of techniques used to grow and study biofilms. This review intends to remedy this, by giving a critical perspective, highlighting the advantages and limitations of several methods. Accordingly, this review aims at helping scientists in finding the most appropriate and up-to-date methods to study their biofilms. ARTICLE HISTORY

Quinolone antibiotics represent one of the most important classes of anti-infective agents and, although still clinically valuable, their use has been compromised by the increasing emergence of resistant strains, which has become a prevalent clinical problem. Quinolones act by inhibiting the activity of DNA gyrase and topoisomerase IV - two essential bacterial enzymes that modulate the chromosomal supercoiling required for critical nucleic acid processes. The acquisition of quinolone resistance is recognized to be multifactorial and complex. The main resistance mechanism consists of one or a combination of target-site gene mutations that alter the drug-binding affinity of target enzymes. However, other mechanisms such as mutations that lead to reduced intracellular drug concentrations, by either decreased uptake or increased efflux, and plasmid-encoded resistance genes producing either target protection proteins, drug-modifying enzymes or multidrug efflux pumps are known to contribute additively to quinolone resistance. The understanding of these different resistance mechanisms has improved significantly in recent years; however, many details remain to be clarified and the contribution of less-studied mechanisms still needs to be better elucidated in order to fully understand this phenotype.

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Saccharomyces cerevisiae plays a primordial role in alcoholic fermentation and has a vast worldwide application in the production of fuel-ethanol, food and beverages. The dominance of S. cerevisiae over other microbial species during alcoholic fermentations has been traditionally ascribed to its higher ethanol tolerance. However, recent studies suggested that other phenomena, such as microbial interactions mediated by killer-like toxins, might play an important role. Here we show that S. cerevisiae secretes antimicrobial peptides (AMPs) during alcoholic fermentation that are active against a wide variety of wine-related yeasts (e.g. Dekkera bruxellensis) and bacteria (e.g. Oenococcus oeni). Mass spectrometry analyses revealed that these AMPs correspond to fragments of the S. cerevisiae glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein. The involvement of GAPDH-derived peptides in wine microbial interactions was further sustained by results obtained in mixed cultures performed with S. cerevisiae single mutants deleted in each of the GAPDH codifying genes (TDH1-3) and also with a S. cerevisiae mutant deleted in the YCA1 gene, which codifies the apoptosis-involved enzyme metacaspase. These findings are discussed in the context of wine microbial interactions, biopreservation potential and the role of GAPDH in the defence system of S. cerevisiae.

Benzalkonium chloride (BC) is a commonly used disinfectant and preservative. This study describes changes in expression level at the transcriptomic and proteomic level for Escherichia coli K-12 gradually adapted to a tolerance level to BC of 7-8 times the initial MIC. Results from DNA arrays and two-dimensional gel electrophoresis for global gene and protein expression studies were confirmed by real-time quantitative PCR. Peptide mass fingerprinting by MALDI-TOF MS was used to identify differentially expressed proteins. Changes in expression level in adapted cells were shown for porins, drug transporters, glycolytic enzymes, ribosomal subunits and several genes and proteins involved in protection against oxidative stress and antibiotics. Adapted strains showed increased tolerance to several antibiotics. In conclusion, E. coli K-12 adapted to higher tolerance to BC acquired several general resistance mechanisms, including responses normally related to the multiple antibiotic resistance (Mar) regulon and protection against oxidative stress. The results revealed that BC treatment might result in superoxide stress in E. coli. INTRODUCTIONQuaternary ammonium compounds (QACs) such as benzalkonium chloride (BC) and cetrimide are frequently used for disinfection and preservation. QACs are relatively stable, non-corrosive compounds with low toxicity and efficacy over a wide pH range. The destructive mechanism of QACs against bacteria is still not known in detail, but is thought to involve a general perturbation of the lipid bilayer in bacterial membranes. This leads to leakage of cytoplasmic material, damaging and ultimately killing the bacterial cell. For a review see Gilbert & Moore (2005). The QACs have been actively deployed since the 1930s and one has not seen a resistance development similar to that seen for antibiotics in the same period. Still, there are numerous reports of QAC resistance (Heir et al., 1999;Langsrud et al., 2003;Wright & Gilbert, 1987), most often associated with acquisition, or hyperexpression, of multi-drug efflux pumps (Li & Nikaido, 2004;Tikhonova & Zgurskaya, 2004), which has also been associated with changes in MIC of therapeutically important antibiotics serving as substrates to such pumps (Langsrud et al., 2004;Poole, 2004Poole, , 2005. Because of their relatively impermeable outer membrane, Gram-negative bacteria are intrinsically more resistant to QACs than Gram-positives (McDonnell & Russell, 1999), but both Gram-negative and Gram-positive BC-resistant bacteria have been isolated from the food industry (Aase et al., 2000;Heir et al., 1995;Langsrud et al., 2003;Soumet et al., 2005).Several studies have linked intrinsic/natural resistance of Gram-negative bacteria to tenside-based disinfectants, such as QACs, to the low permeability of the outer membrane, or broad-spectrum efflux systems (Denyer & Maillard, 2002;Nikaido, 2001). Acquisition of resistance in Escherichia coli has been related mainly to changes in the composition of lipopolysaccharide (LPS) and fatty acids in the membrane (...

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Listeria monocytogenes is the causative agent of listeriosis, one of the most significant foodborne diseases in industrialized countries. The complete genome of the L. monocytogenes EGDe strain, belonging to the serogroup 1/2a, has been sequenced and is comprised of 2853 open reading frames. The objective of the current study was to construct a two-dimensional (2-D) database of the proteome of this strain. The soluble protein fractions of the microorganism were recovered either in the mid-log or in the stationary phase of growth at 37 degrees C. These fractions were analyzed by 2-D electrophoresis (2-DE), using immobilized pH gradient strips of various pH values (3-10, 3-6, and 5-8) for the first-dimensional separations and 12.5% acrylamide gels for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 201 protein spots corresponding to 126 different proteins were identified by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The 2-DE maps presented here provide a first basis for further investigations of protein expression in L. monocytogenes. In this way, the comparison of proteome between cells in the exponential or stationary phase of growth at 37 degrees C allowed us to characterize 161 variations in protein spot intensity, of which 38 were identified. Among the differentially expressed proteins were ribosomal proteins (RpsF, RplJ, and RpmE), proteins involved in cellular metabolism (GlpD, PdhD, Pgm, Lmo1372, Lmo2696, and Lmo2743) or in stress adaptation (GroES and ferritin), a fructose-specific phosphotransferase enzyme IIB (Lmo0399) and different post-translational modified forms of listeriolysin (LLO).

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