Part I RisksRisks
3.1 Introduction: biofilm formation and detection
This chapter deals with biofilm formation, sampling and detection methods, pathogens in biofilms, persistent and non-persistent microbes, prevention of biofilm formation and biofilm removal as well as future trends in biofilm control in the food industry. Microbes that inhabit contact and environmental sites in food processing are mostly harmful because microbial communities in the wrong places lead to contamination of surfaces and of the product produced in the process (Wirtanen, 1995). Documented biofilms have been almost entirely composed of bacteria, and the types of bacterial biofilms particularly related to pathogens are detailed in Section 3.2. There are, however, very few published studies concerning yeast biofilms in food processing. StorgaÊrds et al. (1997) studied the tendency of spoilage yeasts isolated from brewery samples to form biofilms. This study showed that the slow-growing strains covered tested surfaces with 2±4% biofilm in 10 days; fast biofilm producers had already covered the whole surface in 2 days. In addition to the problems in food industry, biofilm formation also causes problems in food-related systems, e.g. industrial water systems as well as the paper and packaging industry (Bryers, 2000; Alakomi et al., 2002). On the positive side, however, biofilms have also been applied successively in food-related processes, e.g. in brewing and in water treatment (KronloÈf, 1994;
Zottola and Sasahara, 1994; Wong and Cerf, 1995; Bryers, 2000).
3.1.1 Factors affecting biofilm formation
In order to be able to survive hostile environmental factors such as heat and chemicals, microbes in microcolonies have a tendency to form protective
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Biofilm risks
G. Wirtanen and S. Salo, VTT Biotechnology, Finland
extracellular matrices, which mainly consist of polysaccharides and glyco-proteins, and are called biofilms (Wirtanen, 1995). The microcolony formation is the first stage in biofilm formation, which occurs under suitable conditions on any surface ± both inert and living. Microbes can start up this formation when there is water or moisture available (Bryers, 2000). Physical parameters such as fluid flow rate, charge, hydrophobicity and micro-topography of the surface material affect the attachment of cells to the surface. Cells must overcome the energy-intensive repulsion barrier, which affects the particle surfaces (van Loosdrecht et al., 1989). Bacteria with pili could conceivably overcome this barrier to achieve micro-colonisation and biofilm formation (Zottola &
Sasahara, 1994). It has been found that temperatures below 50 ëC promote biofilm formation (Miller & Bott, 1982).
In the food industry, equipment design plays the most important role in combating biofilm formations. The choice of materials and their surface treatments as well as roughness, e.g. grinding and polishing, are important factors for inhibiting the formation of biofilm and making surfaces easier to clean. Treating surface materials so that they reject biofilms can be performed actively to remove or passively to retard biofilm reoccurrence. The cleanliness of surfaces, training of personnel and good manufacturing and design practices are the most important tools in combating biofilm problems in the food industry (Holah & Timperley, 1999; Wirtanen, 2002).
3.1.2 Biofilm formation on food processing surfaces
It is also important to remember that about 85±96% of a biofilm consists of water, which means that only 2±5% of the total biofilm volume is detectable on dry surfaces (Costerton et al., 1981). Biofilm can generally be produced by any microbes under suitable conditions, although some microbes naturally have a higher tendency to produce biofilm than others. A biofilm consists of microbial cell clusters with a network of internal channels or voids in the extracellular polysaccharide and glycoprotein matrix (Carpentier & Cerf, 1993). This allows nutrients and oxygen to be transported from the bulk liquid to the cells (Stoodley et al., 1994; KostyaÂl, 1998).
It has been suggested that the mechanisms of microbial attachment and biofilm build-up occur in two-, three-, five- and eight-step processes (Wirtanen, 1995; Bryers, 2000). The two-step process is divided into reversible and irreversible biofilm formation. The reversible phase involves the association of cells near to but not in contact with the surface. Cells associated with the surface synthesise exopolymers, which irreversibly bind the cells to the surface.
Characklis (1981) described biofilm build-up using the following five steps:
transportation of cells to a wetted surface, absorption of the cells into a conditioning film, adhesion of microbial cells to the wetted surface, reaction of the cells in the biofilm and detachment of biofilm from the surface. Bryers and Weightman (1995) divided the biofilm build-up into the following eight steps:
preconditioning of the surface by macromolecules, transport of cells to the Biofilm risks 47
surface, reversible and irreversible adsorption to the surface, cell replication, transport of nutrients and metabolism, production of extracellular polymers and, finally, detachment.
3.1.3 Sampling and detection of biofilm formation in food processing sites Methods for studying biofilm formation include microbiological, chemical, microscopical and molecular biological methods (Wirtanen, 1995; Holah &
Timperley, 1999; Wirtanen et al., 2000a,b; Salo et al., 2000, 2002; Maukonen et al., 2003). Practical methods for assessing microbes and organic soil on processing surfaces are needed to establish the optimal cleaning frequency of the equipment. Hygiene monitoring is currently based on conventional cultivation using swabbing, rinsing or contact plates. Surface sampling can be improved by wetting the surface in advance. In methods that use swabs, sponges or something similar, the detachment of surface-bound microbes is a limiting factor. In the cultivation of biofilm microbes, it is important for the sample to be detached and mixed properly. Agitation used too forcefully in the detachment of the biofilm from the surface may harm the cells, making them unable to grow on the agar plates, whereas insufficient mixing may result in clumps and inaccurate results.
Ultrasonics detaches about ten times the number of cells from the surface compared with swabbing (Wirtanen et al., 2000b). In biofilm detection the planktonic cell counts of processing fluids should be interpreted with caution because they are not always representative of the sessile organisms found on surfaces, especially in badly designed equipment and process lines. Organisms from extreme environments are difficult to culture and therefore standard plate counts do not give accurate estimates. The choice of agar and incubation conditions during the cultivation is governed by the characteristics of the microbes that are considered to be the most important.
Conventional culturing techniques are used to measure the number of viable cells able to grow on the chosen agar at given circumstances. The plates and slides are usually incubated at 25±30 ëC for 2±3 days. The agars are either nutrient agars, which may contain tryptose, yeast, glucose and agar-agar, or selective agars based on growth inhibitors, e.g. nutritional, antibiotic or acidic compounds. The international standard methods for the detection and enumeration of spoilage and pathogenic microbes are based on culturing techniques (van Netten & Kramer, 1992; Salo et al., 2000).
Impedance techniques can be used to enumerate microorganisms directly on surfaces as the increase in conductance and capacitance due to the metabolic activity of the microbes in the sample leads to a decrease in the impedance. The measurement of the change in impedance value at suitable time intervals provides an impedance curve and thus the detection time of microbial growth in the sample (Firstenberg-Eden, 1986). The detection time depends on the number of microbes in the sample. Results are achieved more rapidly with impedance measurements than with cultivation. Impedance measurement is used in the food industry to control product quality and to assess the effect of 48 Handbook of hygiene control in the food industry
cleaning agents and disinfectants (Holah et al., 1990; Flint et al., 1997;
Wirtanen et al., 1997).
The chemical methods used in the assessment of biofilm formation are indirect methods based on the utilisation or production of specific compounds, e.g. organic carbon, oxygen, polysaccharides and proteins, or on the biofilm microbial activity, e.g. living cells and ATP (adenosine 50-triphosphate) content (Characklis et al., 1982). ATP measurement is a luminescence method based on the luciferine±luciferase reaction. The ATP content of the biofilm is proportional to the number of living cells in the biofilm and provides information about their metabolic activity. Kinetic data obtained for freely suspended cells should not be used to assess immobilised biomass growth, e.g.
biofilm. The ATP method is insensitive and therefore not suitable for hygiene measurements in equipment where absolute sterility is needed, because with most of the reagents used today a count of at least 103bacterial cells is needed to obtain a reliable ATP value (Wirtanen, 1995; Lappalainen et al., 2000).
Important tools in modern biotechnology-related research are based on microscopical techniques. One advantage of microscopical analysis is that it can measure surface-adhered cells, rather than cells that have been detached from the surface. Various microscopical techniques for studying cell adhesion and biofilm formation on surface materials are available including: epifluorescence, scanning and transmission electron microscopy, Fourier transformation infrared spectrometry, quartz-crystal microbalance and infrared spectroscopy as well as confocal laser scanning and atomic force microscopying techniques. Fluorescence is a type of luminescence in which light is emitted from molecules for a short period of time following the absorption of light. Fluorescence occurs when an excited electron returns to a lower-energy orbit and emits a photon of light. Many different fluorochromes have been used for the staining of microbes in food samples, biofilms and environmental samples (Wirtanen, 1995; KostyaÂl, 1998).
Flow cytometry using fluorescent probes is a direct optical technique for the measurement of functional and structural properties of individual cells in a cell population. The cells are forced to flow in single file along a rapidly moving fluid stream through a powerful light source. This technique has been used to determine the viability of protozoa, fungi and bacteria. It measures the viability of a statistically significant number of organisms (5000±25 000 cells per sample). The advantages of flow cytometry are accuracy, speed, sensitivity and reproducibility (Wirtanen et al., 2000b).
In the food industry, the first step is to identify the biofilm problems in a particular process or site. Subsequently, it is important to use the best possible methods for isolation and detection of the biofilm for further characterisation in the laboratory using molecular biology and biochemical methods. These methods can be utilised in the detection and identification of microbes in two ways by performing identification either directly from sample material or indirectly from pure cultures obtained from the samples. The two major techniques applied in the molecular detection and identification of bacteria are the polymerase chain reaction and the hybridisation technique (Maukonen et al., 2003).
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