Phenotypic classification of biofilm formation capacity and flagellar motility according to the presence of specific genes.
Biofilm is characterized by a bacterial population firmly adhered to a surface involved by a self-produced matrix of extracellular polymeric substance. These communities provide longer survival and resistance to adverse conditions such as presence of antibiotics and disinfectants. Various foodborne microorganisms are capable of forming such structures, including Salmonella and Campylobacter, which are the major contaminants at the poultry industry. This biomass can affect the water transport system and pipes, and once the agent is established at the industry, it can form biofilms in any processing area. There are intrinsic and extrinsic mechanisms, and also molecular aspects involved in the biofilm formation. The adoption of several strategies may exhibit effectiveness to prevent the cell adhesion, such as the use of surfaces resistant to biofilm formation. In case of preexisting biofilms, there are physical, chemical, and biological methods used to control and eliminate them. Nanotechnology has emerged as another effective measure as nanometals affect the essential activities of microorganisms. These findings highlight the difficulty in controlling biofilms, due to the strategies used by these agents to adapt and survive in sessile form, causing recurring contamination throughout the poultry chain production, deterioration in the final product and infections in the human host.
- public health
- microbial adhesion
- anti-biofilm agents
The industry destined to food production has a great challenge to maintain the safety of the products to be marketed. Among these challenges, there are the failures that may occur during the cleaning process, which may favor the permanence of microorganisms that are able to form biofilms. Several microorganisms transmitted by food are able to form these structures, among them stands out gender representatives
Biofilms are defined as a process of bacterial cells adhesion to a living or inert surface. These cells clump together forming bacterial communities, which are surrounded by a polymer matrix composed mainly by polysaccharides, as well as proteins and nucleic acids [2, 3]. This extracellular matrix promotes the biofilm protection, inhibiting access of biocidal agents, concentrating nutrients, and preventing dehydration .
Typically, biofilms consist of microorganisms in mixed cultures under symbiotic conditions, which are considered more resistant to chemical agents commonly used for cleaning and sanitizing, as well as to other harsh conditions such as refrigeration, acidity, saltiness, and antibiotics [5–7].
The broiler slaughter industry generates residue rich in protein and lipids, which are deposited on surfaces , favoring the formation of biofilms of these pathogens responsible for frequent public health problems. Thus, these bacteria end up becoming a potential source of contamination within the industry that can be transferred to food or to their packaging, becoming a constant threat of recontamination .
Biofilms cause economic losses due to food spoilage and damage to equipment by biocorrosion, and also for damages caused in humans arising from foodborne infections .
The infections by
There are intrinsic and extrinsic mechanisms involved in the formation of these communities, such as the material, type, and shape of the surface, the electric charge, hydrophobicity, and hydrodynamics. There are also different characteristics that determine the maturity of the biofilm, including the environment, mobility, growth rate, the capacity of cell signaling, and the production of extracellular polymer matrix . Besides these, the molecular aspects are also extremely important, as the presence of
The biofilm maturation allows the development of a primitive homeostasis and circulatory system, with exchange of genetic material and metabolic cooperation coordinated by
About the problems involved in the presence of biofilms, preventing their development and their elimination represents greater security to the produced food and consumer. The adoption of several strategies may exhibit effectiveness in eliminating the use of more resistant surfaces for biofilm formation . In the prevention and in cases of previous formed biofilms, physical, chemical, and biological methods can be used, being the combination of the three methods considered most effective . Nanotechnology has emerged as another alternative as nanometals affect the essential activities of
This data reinforces the difficulty in
2. Characteristics of biofilms
Biofilms are formed by an aggregation of bacteria that adhere to each other and secrete extracellular polymeric substances (EPS), as a pellicle formed in the air-liquid interface. These structures are established in response to various environmental conditions and can be developed by multiple signaling strategies. They are usually composed of a mixture of different species of bacteria .
There are five stages that make up the cycle of formation of these structures: (a) free phase, (b) fixing the surface, (c) microcolony, (d) macrocolony, and (e) dispersion (Figure 1).
The first stage is characterized by reversible fixing facilitated by flagellar motility that allows the range to the surface. This connection is of low intensity and allows full movement of the bacterium, but also allows it to be easily removed by cleaning processes . This weak initial interaction of the bacteria with the substrate involves hydrophobic interactions, electrostatic forces, and van der Waals force, which determine the adhesion between the bacterial cell and the surface .
The second stage is called irreversible fixation, moment when gradual increasing in the bond strength occurs by means of continuous production of exopolymers and adhesins. At this stage, the cell removal from the surface requires the action of mechanical force such as scraping, or by chemical treatment. The most important components for this period are exopolymers, adhesins, and DNA (extracellular DNA with structural function) . This phase involves stronger dipole-dipole, hydrogen bonds, hydrophobic interactions, covalent, and ionic bonds .
The formation of microcolonies is detected in the third stage, which occurs approximately after 2 hours, to
The maturation step is accompanied by the formation of macrocolonies resulting from microbial growth and recruitment of other environmental microorganisms. At this time, diffusion through the matrix of exopolymers is slower than the cellular metabolism and the resulting chemical gradients create microniches. Inside them, the death of bacterial cells is evident in the central region, permitting the formation of cavities where motility is possible for planktonic forms.
In human and animal hosts, the
Outside the host, sessile form of
The behavior of these pathogens in mixed and single cultures differs significantly. Biofilms formed by different species of bacteria, such as those found in food industries, represent a substantial risk, since they can protect each other during the application of chemical agents . It is true that in mixed cultures with
3. Biofilms in poultry production
The absence of water is lethal to the growth of microorganisms, but the existence of a minimum supply of water within the farm may be sufficient for the establishment of biofilm. Drinking fountains are more conducive to bacterial attachment, being the portions covered with rubber with greater biomass, because of the facility to adhere .
The control of biofilm formation in the water distribution systems contribute significantly to improve the health of birds, minimizing the need of antibiotic treatments. All chemicals that are added to the drinking water of the poultry, such as dietary supplements and medicines, settle in biofilms and then may spread and generate residues even after its use has been completed. In that way, cleaning of the drinking fountains of poultry is a practice of extreme importance to be adopted in the farms, to ensure that the chickens receive good quality water . The treatment of water supplied in poultry has gained increasing popularity in Europe , and today it is an essential measure to control pathogens in the poultry production chain.
In order to reduce microbial contamination the producers treat the water that will be destined to the birds with various chemicals such as: chlorine, chlorine dioxide, organic acids, peracetic acid, and hydrogen peroxide. However, these substances have action only under appropriate conditions of temperature and pH  and for a limited period of time, so it should be repeated periodically.
Some producers use chemical control methods in water intended for poultry only once or twice a week, and others limit this kind of processing to the end of the production cycle, i.e., there is a lack of standardization and proper methodology that undertake control efficiency and reduction of pathogens in the water, and potentiates the transmission of pathogens by water .
Studies have shown that one of the most common sources of contamination are from biofilms developed inside the water supply pipe, where a variety of microorganisms proliferate surrounded by mud, and are adhered to surface and continuously release planktonic cells in water .
Ventilation systems (coolers) are also a favorable area for microbial aggregation, especially in situations where there is a preexisting biofilm .
Higher biofilm formation rates are observed at the chicken processing steps because of large amount of moisture in the environment. Several critical points are identified during production, such as plastic curtains, mats, scalding tanks, chiller, and stainless steel tools .
A research with
The fact that the chicken reaches the abattoir harboring pathogens such as
The survival of
Thus, it must consider that proper control of cleaning methods used in the poultry production system is of paramount importance and should include strict compliance with the established biosecurity protocols. This applies mainly to difficult decontamination environments such as feed mills, agricultural environments, and farms [41, 42].
4. Intrinsic mechanisms of
For the biofilm establishment, both environmental variations and the microorganism itself correlate with genes that are expressed by bacteria in sessile form.
Table 1 illustrates some of the molecular mechanisms involved directly in biofilm formation and the link between this mechanism and the flagellar apparatus.
The several genes that encode different flagellar proteins clearly show the necessity of flagella on biofilm formation in
The lack of genes involved in flagella expression activation, as well as those that are involved in chemotaxis (
The biofilm matrix is composed basically by exopolysaccharides (EPS), proteins and DNA and thus the regulatory genes of these molecules production and the availability of nutrients define quantitative variations in this composition .
In addition to the flagella, the genes that decisively affect biofilm formation in
The stress response generates metabolic changes in the expression of nearly the totality of the bacterial genome. Both, increasing the response to stress protein expression and decreasing of metabolic activity express the conditions of sessile microorganism and contributes to tolerance to the harsh conditions that bacteria are submit in biofilms. Iron uptake and stress response proteins are highly expressed in biofilms. Metabolic protein expression will vary, depending on the need, suggesting that distinct changes in metabolism mark the transition between modes of planktonic and sessile growth [47, 48].
|Flagella adhesion||Larger flagellin||+||+|
|Flagellin homologous, adhesion||+||=|
|Flagellar sigma factor||+||=|
|Flagellar proteic secretor apparatus||+||+|
|Binfunctional synthetase II||−||=|
|Poly-P kinase 1||−||=|
|Poly-P kinase 2||−||=|
|Oxidative stress regulator||+||=|
|Thermic stress regulator||+||+|
|Metabolic growth regulator||+||=|
|Osmotic shock regulator||+||=|
5. Intrinsic mechanisms of
Even in the case of intrinsic mechanisms of
Among the most important genes associated with biofilm formation, there is the
6. Extrinsic factors linked to biofilms
About the environmental conditions, there are many factors that determine the production, development, and maintenance of biofilms, including the pH, temperature, type of material and the surface roughness, the presence of organic and inorganic compounds, the condition of dynamic flow, osmotic pressure, oxygen concentration, concentration and bioavailability of nutrients, and the presence of antimicrobial agents in the medium. This is due to the fact that different environmental conditions will generate different responses in gene regulations of the bacteria and thus the behavior of biofilms .
The sessile mode of growth is enhanced in low quantity of nutrient conditions. This fact is noted by elevated LPS production in the matrix. An example is related to the use of excessive nutritive media, such as Bolton, Brucella, and Brain Heart Infusion broths,
The role of temperature in the formation of biofilms is more complex, varies among species and the changes are related to other environmental conditions .
The physicochemical properties of the surface exert a strong influence on the adherence of microorganisms. In general, the bacteria adhere more easily to the hydrophobic surfaces like plastics, than hydrophilic surfaces such as glass or metal .
The osmotic stress inhibits biofilm formation and leads to dispersion of the existing structure. The addition of NaCl (sodium chloride), glucose, or sucrose significantly decreases the formation of biofilm on
The effect of oxygen tension in biofilm formation of
7. The importance of the
8. Biofilm control
Given the biofilms resistance of
The equipment design and the choice of the materials and coatings used in the food industry are extremely important in preventing biofilm formation. This is because even adopting the most effective cleaning and sanitizing programs, it is not possible to compensate for problems caused by faulty equipment, which have inaccessible corners, cracks, crevices, valves, and joints, which are vulnerable points for biofilm accumulation .
The use of well-designed equipment associated with the adoption of effective hygiene measures allow the removal of unwanted material from surfaces, including microorganisms, foreign materials, and residues from cleaning products [63, 64].
New technologies for detecting the presence of biofilms have been developed in order to control the colonization of surfaces by bacteria and identify the early stages of biofilm formation and development . A research performed by Ref.  developed a mechatronic sensor to surface capable of providing various information such as the presence of biofilms in the early stages, presence of cleaning products in the surface, and differentiation of the type of cleaning employed (biological or chemical).
Once the biofilm is already established, it should be emphasized cleaning processes using mechanical action, which is one of the main measures for their elimination or control , because the friction acts on the matrix disruption, exposing deeper layers and making the microorganisms more accessible.
Generally, disinfectants do not penetrate the biofilm matrix after an inefficient cleaning procedure and, therefore, does not destroy all the biofilm cells , reaching only the outer layers. Cleaning is the first step and very important to improve the sanitation of equipment and facilities . It is important to remove effectively the food wastes that may contain microorganisms or promote microbial growth.
The use of high temperature may reduce the need for application of mechanical forces, such as turbulence in the wash water. The chemicals commonly used for cleaning are surfactants or alkalis, used to suspend and dissolve the food residues by reducing the surface tension, emulsify fats, and denature proteins .
In addition to the mechanical action, other measures must be taken to prevent and control microbial adhesion. In this sense, the facilities, equipment, and utensils should be washed daily and disinfected with the use of microbicides substances previously approved by legislation.
However, there are studies showing that even using the recommended concentration of sanitizing, resistance of bacteria in biofilms still exists. A study performed by Ref.  evaluated the bactericidal capacity of peracetic acid on
The disinfection is the use of products for elimination of microorganisms, especially pathogenic. The purpose of disinfection is to reduce the microbial load remaining on the surface after cleaning and prevents their proliferation before restarting the production process. Disinfectants must be effective, safe, and easy to handle, they should be removed from surfaces easily, using water, leaving no residue in the final product that may affect the consumer .
|Mechanism of action||Examples||Compounds|
|Blocking in the bacterial adhesion||Policides||2-pyridone bicyclic|
|Iron chelators||Lactoferrin; plant extracts, tannins|
|Competition for receptor sites/AIs degration||Furanones halogenated and peptide inhibitor of RNA III (RIP)|
|Mature Biofilms—Matriz||Enzymes||Proteinase, typsin, DNAase, sodium|
|Alteraçāo no pH||Detergentes ácidos/alcalinos|
|Mature Biofilms- Biomass||Nanoparticles||Zinc, silver, titanium, gold.|
The chemicals currently used in disinfection processes belong to the following types: acidic compounds, biocides, aldehyde-based, caustics, chlorine, hydrogen peroxide, iodine, isothiazolinones, ozone, peracetic acid, phenols, biguanides, and surfactants [64, 69]. Some examples of agents that may be used to control and/or eliminate biofilms of
The strategies most used in industry involve the removal of biofilms already installed, by removing the matrix and/or bacterial biomass. As a first step is quoted to use hygienic processes with enzymatic detergents and compounds that promote the sudden change in pH and subsequent matrix liquefaction .
The use of enzyme-based detergents may be useful to improve the cleaning process. However, due to the heterogeneity in biofilm matrices, it is necessary to know the exact composition for which suitable enzymatic treatments can be applied , so that a mixture of different enzymes can increase the spectrum action on biofilm degradation. These enzymatic processes have the advantage of disaggregate biofilm agglomerates, rather than just remove them from the surface, as is the case of mechanical action.
Another important point to be analyzed for the elimination of bacteria in mature biofilm is the involvement of strain-dependent characteristics, since there are molecular intrinsic factors that may act by preventing the effectiveness of the agents, hindering its penetration depending on the composition of the matrix, and also the mechanism of action of the applied agent.
In general, the policides act by inhibiting adhesin and essential fimbriae synthesis in the process of fixing the bacteria to surfaces. The iron chelating agents prevent the availability of this element in the initial process of accession, essential for the biofilm formation. Inactivation of the
The surfactants and biosurfactants are also alternatives that can be used in combating biofilm formation. A study of  reported that pretreated surfaces with surfactants may have potential higher than 90% in the prevention of bacterial adhesion, and biosurfactants such as rhamnolipids and short chain fatty acids can promote rupture on biofilms [73, 74]. Since surfactin from
The nanoparticles, as well as the antimicrobial peptides (AMPs), appears as a current strategy for the removal of biomass of biofilms, since they are stable at high temperature and pressures, have inactivates potential, can easily penetrate the matrix, are less likely to develop resistance, have minimal effect on the human cells and can be used to extend the shelf life of fresh and meat products [76, 77].
Combinations of different treatments, with different types of actions are also useful. For example, ultrasound waves  were associated with the improvement performance of proteolytic enzymes. These processes target the biofilm matrix, causing the disaggregation and dispersion of the biomass. However, they are not efficient in eliminating these microorganisms, which can adhere to the surface again and restart a new cycle of the biofilm formation.
Alternatively, under increasing interest for biofilm control is to use bacteriophages, which are viruses with high specificity that infect and lyse bacteria and diffuse easily into the matrix layers, including in mature biofilms [79–81]. This technology is still in development, so information about the bacteriophage action in biofilms is still scarce . However, it is known that the infection of biofilm by a phage depends on their chemical composition and also environmental factors such as temperature, growth phase, media, and phage concentration .
Studies on the use of natural antimicrobials as antibiofilm substances, for example, compounds extracted from aromatic plants , which are recognized as safe for not leave toxic residues for the consumer and does not change the quality of final product. These compounds have demonstrated their antimicrobial activity in planktonic bacteria and some is being evaluated for its potential in eradicating biofilms.
A research performed by Ref.  tested the influence of carvacrol, a broad spectrum antimicrobial found in essential oils of herbs such as oregano and thyme, on biofilm of
The use of combined actions involving two or more types of chemical, physical, and natural treatments have been reported as the measure of control with more effectiveness against biofilm formation . These treatments can synergistically enhance and broaden the spectrum of actions to eradicate biofilms.
Despite several options for new treatments to prevent and remove biofilms, further studies need to be carried out continuously to understand the dynamics of these structures.
Whereas biofilms are constant sources of contamination of production systems for spoilage and pathogens, having economic and public health impacts, prevention should be included in the objectives of the quality of industrial controls. Among the actions required in all strategies, should be included the frequent monitoring, and internal policies to ensure compliance with the preestablished hygiene plans, particularly, respecting the intervals between cleaning processes.
To Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) for the financial support.