Abstract: Biofouling of nanofiltration (NF) and reverse osmosis (RO) membranes for water treatment has been the subject of increased research effort in recent years. A prerequisite for undertaking fundamental experimental investigation on NF and RO processes is a procedure called compaction. This involves an initial phase of clean water permeation at high pressures until a stable permeate flux is reached. However water quality used during the compaction process may vary from one laboratory to another. The aim of this study was to investigate the impact of laboratory water quality during compaction of NF membranes. A second objective was to investigate if the water quality used during compaction influences initial bacterial adhesion.
Experiments were undertaken with NF270 membranes at 15 bar for permeate volumes of 0.5L, 2L, and 5L using MilliQ, deionized or tap water. Membrane autopsies were performed at each permeation point for membrane surface characterisation by contact angle measurements, profilometry, and scanning electron microscopy. The biological content of compacted membranes was assessed by direct epi-fluorescence observation following nucleic acid staining. The compacted membranes were also employed as substrata for monitoring the initial adhesion of Ps. fluorescens under dynamic flow conditions for 30 minutes at 5 minutes intervals.
Compared to MilliQ water, membrane compaction using deionized and tap water led to decreases in permeate flux, increase in surface hydrophobicity and led to significant build-up of a homogenous fouling layer composed of both living and dead organisms (>106 cells.cm-2). Subsequent measurements of bacterial adhesion resulted in cell loadings of 0.2×105, 1.0×105 cells×cm-2 and 2.6×105 cells.cm-2 for deionized, tap water and MilliQ water, respectively. These differences in initial cell adhesion rates demonstrate that choice of laboratory water can significantly impact the results of bacterial adhesion on NF membranes. Standardized protocols are therefore needed for the fundamental studies of bacterial adhesion and biofouling formation on NF and RO membrane. This can be implemented by first employing pure water during all membrane compaction procedures and for the modelled feed solutions used in the experiment.
Abstract: Staphylococcus aureus is a pathogenic bacterium capable of developing biofilms on food-processing surfaces, a pathway leading to cross contamination of foods. The purpose of this study was to investigate the influence of environmental stress factors found during seafood production on the adhesion and biofilm-forming properties of S. aureus. Adhesion and biofilm assays were performed on 26 S. aureus isolated from seafood and two S. aureus reference strains (ATCC 6538 and ATCC 43300). Cell surface properties were evaluated by affinity measurements to solvents in a partitioning test, while adhesion and biofilm assays were performed in polystyrene microplates under different stress conditions of temperature, osmolarity, and nutrient content. The expression of genes implicated in the regulation of biofilm formation (icaA, rbf and σ( B )) was analyzed by reverse transcription and quantitative real time PCR. In general, S. aureus isolates showed moderate hydrophobic properties and a marked Lewis-base character. Initial adhesion to polystyrene was positively correlated with the ionic strength of the growth medium. Most of the strains had a higher biofilm production at 37 °C than at 25 °C, promoted by the addition of glucose, whereas NaCl and MgCl(2) had a lower impact markedly affected by incubation temperatures. Principal Component Analysis revealed a considerable variability in adhesion and biofilm-forming properties between S. aureus isolates. Transcriptional analysis also indicated variations in gene expression between three characteristic isolates under different environmental conditions. These results suggested that the prevalence of S. aureus strains on food-processing surfaces is above all conditioned by the ability to adapt to the environmental stress conditions present during food production. These findings are relevant for food safety and may be of importance when choosing the safest environmental conditions and material during processing, packaging, and storage of seafood products.
Abstract: Diffusion of entities inside biofilm triggers most mechanisms involved in biofilm-specific phenotypes. Using genetically engineered hydrophilic and hydrophobic cells of Lactococcus lactis yielding similar biofilm architectures, we demonstrated by fluorescence correlation spectroscopy that bacterial surface properties affect diffusion of nanoparticles through the biofilm matrix.
Abstract: Several studies have shown that consumers may not clean cutting boards properly between preparation of raw and cooked meat. Cutting boards may therefore act as sources for contamination of cooked meat or other ready-to-eat foods with pathogenic and spoilage bacteria. The aim of the work was to investigate if cutting boards containing the antimicrobial compound triclosan can reduce the viability of bacteria, thus acting as a hygiene barrier. Survival and growth of food pathogens and spoilage bacteria on two cutting boards without antimicrobials and a commercial cutting board containing triclosan were tested. No difference in bacterial counts on cutting boards without and with triclosan was found after exposure to naturally contaminated chicken filets for one hour. Pathogenic and spoilage bacteria were inoculated on coupons (6.7-7 log per coupon) of cutting boards and incubated at 25°C at controlled relative humidity for 24 and 72 h. At a relative humidity of 100%, growth of Escherichia coli, Salmonella, Staphylococcus aureus, coagulase-negative staphylococci (CNS) and Serrratia spp. was observed and no antibacterial effect of the triclosan-containing board was found except for against Listeria monocytogenes. At lower humidity (70% RH) less growth was found on the triclosan-containing cutting board than untreated boards after 24h. After 72 h of incubation, cell counts were reduced on triclosan-containing boards, with the most pronounced antibacterial effects observed against Salmonella, S. aureus and CNS. For S. aureus and Salmonella it was found that when a lower initial cell count was applied (3.5 log per coupon), the triclosan-containing board had an antibacterial effect under humid conditions, as well as a more pronounced antibacterial effect under dry conditions. An agar overlay assay showed that triclosan migrated out of the coupons. Repeated washing of the triclosan-containing cutting boards reduced the antibacterial effect, thus the amount of triclosan available on the surface seemed to be limited. In conclusion, using triclosan-containing cutting boards as a hygienic barrier may only work under certain conditions (low humidity, long exposure time, and clean conditions) and not against all genera of bacteria.
Abstract: Surfaces in industrial settings provide a home for resident biofilms that are likely to interact with the attachment, growth and survival of pathogens such as Listeria monocytogenes. Experimental results have indicated that L. monocytogenes cells were inhibited by the presence of a model resident flora (Lactococcus lactis) in dual-species continuous flow-biofilms, and are spatially restricted to the lower biofilm layers. Using a new, simplified individual-based model (IBM) that simulates bacterial cell growth in a three-dimensional space, the spatial arrangements of the two species were reconstructed and their cell counts successfully predicted. This model showed that the difference in generation times between L. monocytogenes and L. lactis cells during the initial stages of dual-species biofilm formation was probably responsible for the species spatialization observed and the subsequent inhibition of growth of the pathogen.
Abstract: Shigatoxin-producing Escherichia coli (STEC) causes severe infections, and has been the cause of a number of foodborne outbreaks. Knowledge on the survival of STEC is crucial in order to limit the risk of cross contamination and transfer of STEC to food during processing. In this study survival of STEC and non-STEC on surfaces under various humidities, temperatures and in the presence of different types of soil was investigated. A model system with controlled relative humidity and temperature was established by using saturated salt solutions. All the 12 STEC strains had a reduction in viable count during incubation at 70% RH at 12 degrees C. The reduction was 2-3.5 log and 4.5-5.5 log after 1 and 7 days of incubation, respectively. Surviving cells were observed after 19 days of incubation. The STEC strains were more resistant to desiccation than non-STEC strains. STEC survived better at 12 degrees C, compared to 20 degrees C. The survival of STEC was much lower than the survival of a Staphylococcus simulans strain tested, which showed less than 1 log reduction until day 7 at 70% RH at 12 degrees C, while several STEC strains had comparable survival to a Salmonella Agona strain. The survival of two STEC strains tested was highest at 98% RH. The lowest survival was observed at 85% RH, with better survival at drier conditions. Presence of proteins and glucose protected the cells at dry conditions. Two commercial disinfectants tested at in-use concentration had limited effect (0.8-2.5 log reduction) against STEC on stainless steel, especially for cells incubated at high relative humidity (98% RH). STEC surviving on surfaces in the food industry may impose a risk for cross contamination. Cleaning and use of suitable disinfectants will reduce the survival of STEC, but surfaces should be allowed to dry completely since humid conditions will promote the survival and growth of STEC.
Abstract: A meat factory commensal bacterium, Acinetobacter calcoaceticus, affected the spatial distribution of Escherichia coli O157:H7 surface colonization. The biovolume of E. coli O157:H7 was 400-fold higher (1.2 x 10(6) microm(3)) in a dynamic cocultured biofilm than in a monoculture (3.0 x 10(3) microm(3)), and E. coli O157:H7 colonized spaces between A. calcoaceticus cell clusters.
Abstract: The presence of Salmonella enterica serovars in feed ingredients, products and processing facilities is a well recognized problem worldwide. In Norwegian feed factories, strict control measures are implemented to avoid establishment and spreading of Salmonella throughout the processing chain. There is limited knowledge on the presence and survival of the resident microflora in feed production plants. Information on interactions between Salmonella and other bacteria in feed production plants and how they affect survival and biofilm formation of Salmonella is also limited. The aim of this study was to identify resident microbiota found in feed production environments, and to compare the survival of resident flora strains and Salmonella to stress factors typically found in feed processing environments. Moreover, the role of dominant resident flora strains in the biofilm development of Salmonella was determined.
Abstract: Planktonic Listeria monocytogenes cells in food-processing environments tend most frequently to adhere to solid surfaces. Under these conditions, they are likely to encounter resident biofilms rather than a raw solid surface. Although metabolic interactions between L. monocytogenes and resident microflora have been widely studied, little is known about the biofilm properties that influence the initial fixation of L. monocytogenes to the biofilm interface. To study these properties, we created a set of model resident Lactococcus lactis biofilms with various architectures, types of matrices, and individual cell surface properties. This was achieved using cell wall mutants that affect bacterial chain formation, exopolysaccharide (EPS) synthesis and surface hydrophobicity. The dynamics of the formation of these biofilm structures were analyzed in flow cell chambers using in situ time course confocal laser scanning microscopy imaging. All the L. lactis biofilms tested reduced the initial immobilization of L. monocytogenes compared to the glass substratum of the flow cell. Significant differences were seen in L. monocytogenes settlement as a function of the genetic background of resident lactococcal biofilm cells. In particular, biofilms of the L. lactis chain-forming mutant resulted in a marked increase in L. monocytogenes settlement, while biofilms of the EPS-secreting mutant efficiently prevented pathogen fixation. These results offer new insights into the role of resident biofilms in governing the settlement of pathogens on food chain surfaces and could be of relevance in the field of food safety controls.
Abstract: Listeria monocytogenes is a food pathogen that can attach on most of the surfaces encountered in the food industry. Biofilms are three-dimensional microbial structures that facilitate the persistence of pathogens on surfaces, their resistance toward antimicrobials, and the final contamination of processed goods. So far, little is known about the structural dynamics of L. monocytogenes biofilm formation and its regulation. The aims of this study were, by combining genetics and time-lapse laser-scanning confocal microscopy (LSCM), (i) to characterize the structural dynamics of L. monocytogenes EGD-e sessile growth in two nutritional environments (with or without a nutrient flow), and (ii) to evaluate the possible role of the L. monocytogenes agr system during biofilm formation by tracking the spatiotemporal fluorescence expression of a green fluorescent protein (GFP) reporter system. In the absence of nutrient flow (static conditions), unstructured biofilms composed of a few layers of cells that covered the substratum were observed. In contrast, when grown under dynamic conditions, L. monocytogenes EGD-e biofilms were highly organized. Indeed, ball-shaped microcolonies were surrounded by a network of knitted chains. The spatiotemporal tracking of fluorescence emitted by the GFP reporter system revealed that agr expression was barely detectable under static conditions, but it progressively increased during 40 h under dynamic conditions. Moreover, spatial analysis revealed that agr was expressed preferentially in cells located outside the microcolonies. Finally, the in-frame deletion of agrA, which encodes a transcriptional regulator, resulted in a decrease in initial adherence without affecting the subsequent biofilm development.
Abstract: The first step in biofilm formation is bacterial attachment to solid surfaces, which is dependent on the cell surface physico-chemical properties. Cell wall anchored proteins (CWAP) are among the known adhesins that confer the adhesive properties to pathogenic Gram-positive bacteria. To investigate the role of CWAP of non-pathogen Gram-positive bacteria in the initial steps of biofilm formation, we evaluated the physico-chemical properties and adhesion to solid surfaces of Lactococcus lactis. To be able to grow in milk this dairy bacterium expresses a cell wall anchored proteinase PrtP for breakdown of milk caseins.
