Biofilm-forming ability and resistance to industrial disinfectants of Staphylococcus aureus isolated from fishery products

Adhesion and biofilm-forming ability of twenty six S. aureus strains previously isolated from fishery products on stainless steel was assessed. All strains reached counts higher than 10⁴ CFU/cm² after 5 h at 25 °C. Most strains also showed a biofilm-forming ability higher than S. aureus ATCC 6538 – reference strain in bactericidal standard tests – by crystal violet staining. In addition, it seems that food-processing could have produced a selective pressure and strains with a high biofilm-forming ability were more likely found in highly handled and processed products.The efficacy of the industrial disinfectants benzalkonium chloride (BAC), sodium hypochlorite (NaClO) and peracetic acid (PAA) against biofilms and planktonic counterparts was also examined in terms of minimum biofilm eradication concentration (MBEC) and minimum bactericidal concentration (MBC), respectively. Biofilms showed an antimicrobial resistance higher than planktonic cells in all cases. However, no correlation was found between MBEC and MBC, likely due to differences in biofilm extracellular matrix (composition, content and architecture) between strains. BAC resistance increased as biofilms aged. Generally, biofilm formation seemed to attenuate the effect of low temperatures on BAC resistance. PAA was found to be most effective against both biofilms and planktonic cells, followed by NaClO and BAC. Resistance did not follow the same order for each biocide, which remarks the need of using a wide collection of strains in standard tests of bactericidal activity to ensure a proper application of disinfectants. Doses recommended by manufacturers for BAC, PAA and NaClO to disinfect food-contact surfaces were lower than doses for complete biofilm removal (i.e. MBEC) under some environmental conditions common in the food industry, which questions bactericidal standard tests and promotes the search for new strategies for biofilm removal.     

The specific anti-biofilm effect of gallic acid on Staphylococcus aureus by regulating the expression of the ica operon

Staphylococcus aureus (S. aureus) biofilms are of considerable interest in food safety because biofilms can increase the risk of food contamination and enhance the pathogenicity of bacteria. The ica-encoded polysaccharide intercellular adhesin (PIA) plays an important role in biofilm formation. In this study, the MIC of gallic acid against S. aureus in suspension and in biofilms was 2 mg/mL and 4 mg/mL, respectively. Quantitative crystal violet staining of biofilms showed that 2 mg/mL gallic acid can effectively inhibit biofilm formation and the ESEM images clearly showed the three-dimensional biofilm morphology of the S. aureus and the resulting anti-biofilm effect. The determination of viable bacteria in the biofilm revealed that gallic acid penetrated the biofilm to kill S. aureus, the bactericidal effect on the biofilm bacteria was comparable to that of planktonic bacteria. We further explored the influence of gallic acid on ica family gene expression and polysaccharide slime formation in S. aureus biofilm formation. The results showed that icaR was significantly activated that; icaA and icaD were downregulated in a dose-dependent manner with increasing concentrations of gallic acid; however, the expression of icaB and icaC was not significantly affected. The polysaccharide slime formation was reduced as well. Based on these results, gallic acid, as a natural substance, may play an important role in the food industry.     

Rutin inhibits mono and multi-species biofilm formation by foodborne drug resistant Escherichia coli and Staphylococcus aureus

Considering the role of biofilm in food spoilage and the food industry, inhibition of biofilm formation by natural agents is expected to be safe and could also enhance the efficacy of other antimicrobial strategies for controlling microbial food spoilage. Plant flavonoids are known for their diverse biological activity including antimicrobial. Therefore, rutin was investigated for its biofilm inhibitory activity at sub-minimum inhibitory concentrations (sub-MICs) values against common foodborne pathogens (Escherichia coli and Staphylococcus aureus). Minimum inhibitory concentrations (MIC) ranged from 400 to 1600 μg/ml against the selected strains. Sub-MICs (1/16 × MIC to 1/2 × MIC) were used to assess the inhibition of biofilm formed by E. coli and S. aureus in microtitre plate assay. Mono strain biofilm formation by Escherichia coli and Staphylococcus aureus was greatly reduced by rutin at their respective 1/2 × MIC. For multi-species (E. coli: and S. aureus) biofilm formation, the reduction in biofilm production was concentration dependent. No significant bacteria mass reduction was recorded for any sub-MIC. SEM images of biofilm inhibition on steel chips confirmed the reduction in number of microcolonies. Exopolysaccharide production responsible for adherence and maturation of biofilms was also significantly (p ≤ 0.05) reduced at respective concentrations of rutin in tested strains. To the best of our knowledge, this is the first study describing the effect of flavonoid, rutin on multi-species biofilms consisting of S. aureus and E. coli. Findings of the study indicate a potential application of rutin in the prevention of biofilm on industrial equipment and food contact surfaces and prevent food contamination and spoilage.     

Effectiveness of a phage cocktail as a biocontrol agent against L. monocytogenes biofilms

Listeria monocytogenes can persist and form biofilms in a food environment which are difficult to eradicate because biofilms are inherently resistant to a variety of antimicrobial treatments. Therefore, alternative approaches such as bacteriophages have been suggested as a promising biocontrol agent against biofilms. The aim of this study was to evaluate the efficacy of a cocktail bacteriophage product (ListShield™) against L. monocytogenes biofilms. These biofilms were established on lettuce, stainless steel, rubber, and a MBEC biofilm device and exposed to the ListShield™ phage preparation (1 × 10⁸ PFU/mL) for 2 h. ListShield™ had sufficient potency to significantly reduce the biofilm (P < 0.05) in all cases. Biofilm reduction achieved after ListShield™ treatment on the stainless steel coupon was 1.9–2.4 log CFU/cm² and on the rubber surface approximately 1.0 log CFU/cm². Phage application on lettuce inactivated biofilm bacteria up to 0.7 log CFU/cm². These results suggest that bacteriophage preparation ListShield™ is an effective tool for the inactivation of L. monocytogenes biofilms in the food industry.     

Adhesion of Staphylococcus aureus on stainless steel treated with three types of milk

Staphylococcus aureus has the ability to adhere and to form biofilm on inert surface such as stainless steel commonly used in food industry. The biofilm formed on the surface of milk processing equipments could be a source of dairy products contamination. This contamination causes a food poisoning. In this paper the S. aureus adhesion on stainless steel treated by three types of milk (ultrahigh-temperature (UHT)-treated milk; UHT skimmed milk, UHT semi-skimmed milk) was investigated.Stainless steel was exposed to three types of milk with a different amount of fat component. Contact angles measurements were used to determine the surface physicochemical properties of substratum treated with the three milk products. The hydrophobicity and electron acceptor properties of stainless steel seem to be decreasing with the amount of fat component present in milk but its electron donor property increase with this component. The ability of S. aureus to adhere to stainless steel treated and untreated with milk was also examined. Treatment with the three types of milk reduces bacterial attachment. On treated substratum, the adhesion extent was affected by the type of milk and consequently by the amount of fat component. The lower and the higher adhesion were obtained when the steel was treated by the UHT semi-skimmed milk and UHT skimmed milk respectively. The correlation between physicochemical properties and S. aureus adhesion show that this latter was controlled by hydrophobicity and electron donor properties.The findings of this work can contribute to develop strategies for prevent S. aureus adhesion on stainless steel and biofilm formation. Also they could be taken into account in cleaning and disinfection procedures.     

Evaluation of rhamnolipid and surfactin to reduce the adhesion and remove biofilms of individual and mixed cultures of food pathogenic bacteria

Biofilms represent a great concern for food industry, since they can be a source of persistent contamination leading to food spoilage and to the transmission of diseases. To avoid the adhesion of bacteria and the formation of biofilms, an alternative is the pre-conditioning of surfaces using biosurfactants, microbial compounds that can modify the physicochemical properties of surfaces changing bacterial interactions and consequently adhesion. Different concentrations of the biosurfactants, surfactin from Bacillus subtilis and rhamnolipids from Pseudomonas aeruginosa, were evaluated to reduce the adhesion and to disrupt biofilms of food-borne pathogenic bacteria. Individual cultures and mixed cultures of Staphylococcus aureus, Listeria monocytogenes and Salmonella Enteritidis were studied using polystyrene as the model surface. The pre-conditioning with surfactin 0.25% reduced by 42.0% the adhesion of L. monocytogenes and S. Enteritidis, whereas the treatment using rhamnolipids 1.0% reduced by 57.8% adhesion of L. monocytogenes and by 67.8% adhesion of S. aureus to polystyrene.Biosurfactants were less effective to avoid adhesion of mixed cultures of the bacteria when compared with individual cultures. After 2 h contact with surfactin at 0.1% concentration, the pre-formed biofilms of S. aureus were reduced by 63.7%, L. monocytogenesby 95.9%, S. Enteritidis by 35.5% and the mixed culture biofilm by 58.5%. The rhamnolipids at 0.25% concentration removed 58.5% the biofilm of S. aureus, 26.5% of L. monocytogenes, 23.0% of S. Enteritidis and 24.0% the mixed culture after 2 h contact. In general, the increase in concentration of biosurfactants and in the time of contact decreased biofilm removal percentage. These results suggest that surfactin and rhamnolipids can be explored to control the attachment and to disrupt biofilms of individual and mixed cultures of the food-borne pathogens.     

Supercritical fluid extracts of Moringa oleifera and their unsaturated fatty acid components inhibit biofilm formation by Staphylococcus aureus

Biofilm formation by Staphylococcus aureus plays a critical role in the persistence of chronic infections because of the ability of the bacterium in biofilms to tolerate antibiotics and host defenses. S. aureus produces hemolysin, which has been implicated in the pathogenesis of sepsis and pneumonia. Hence, the inhibitions of biofilm formation and/or toxin production by S. aureus are viewed as alternative means of addressing infections. In the present study, the antibiofilm activities of Moringa oleifera extracts were examined. Of the various solvent extraction methods examined, the supercritical carbon dioxide extracts of the leaves and seeds of M. oleifera were found to efficiently inhibit biofilm formation by S. aureus. Analyses of the extracts by GC-MS revealed the presences of palmitoleic acid, oleic acid, linoleic acid, linolenic acid, cis-11-eicosenoic acid, and cis-11,14-eicosadienoic acid at concentrations of 0.01% significantly inhibited S. aureus biofilm formation. In addition, supercritical fluid extract of the leaves of M. oleifera and its major component cis-11-eicosenoic acid significantly decreased the hemolysis of human red blood cells by S. aureus. These findings suggest supercritical carbon dioxide fluid extract of M. oleifera and its unsaturated fatty acids are potentially useful for controlling biofilm formation by and the virulence of S. aureus.     

Effects of benzalkonium chloride and ethanol on dual-species biofilms of Serratia liquefaciens S1 and Shewanella putrefaciens S4

Biofilm formation on food-contact surfaces represents a significant risk to public health. To evaluate the effects of chemical disinfections on foodborne biofilms, Serratia liquefaciens S1 and Shewanella putrefaciens S4, which were isolated from the same site in a raw-chicken-processing environment, were selected to establish a dual-species biofilm model. Two disinfectants, benzalkonium chloride (BC) and ethanol, were used to treat mono-species and dual-species biofilms formed by S. liquefaciens S1 and S. putrefaciens S4. The results show that the removal effect of ethanol on the biofilms was more significant than that of BC, but there was no significant difference between the two disinfectants on the killing effect. The S. liquefaciens S1 and S. putrefaciens S4 dual-species biofilms showed stronger resistance to disinfectants than the mono-species biofilms. Moreover, structural observation of biofilms indicates that the extracellular polymeric substance (EPS) plays an important role in the protection of dual-species biofilm.     

Effect of neutral electrolyzed water on lux-tagged Listeria monocytogenes EGDe biofilms adhered to stainless steel and visualization with destructive and non-destructive microscopy techniques

The presence of Listeria monocytogenes in food processing environment is a risk of food contamination by persistent cells due to their ability to attach to stainless steel and other surfaces. We aimed to study biofilms formation of lux-tagged L. monocytogenes EGDe on stainless steel surfaces and their control using neutral electrolyzed water (NEW), where biofilms development was monitored using destructive and non-destructive microscopy techniques. The development of biofilms was monitored for 5 days on stainless steel chips. We used two sources of NEW, commercial (NEW-1) and from a prototype (NEW-2) for treatments of free and biofilm L. monocytogenes EGDe cells. Complete inhibition of L. monocytogenes EGDe free cells was observed after 1 min contact time for both NEW sources, but NEW-1 concentration used (9 mg/L total available chlorine, TAC) was 1.8 times higher. Cells within biofilms were more resistant to NEW compared to planktonic cells. Same concentration of both NEW sources (70 mg/L TAC) exhibited complete inhibition of biofilm cells after 3 min contact time. However, using a sub-lethal dose of 40 mg/L TAC, NEW-2 reduced about 2 log CFU/cm² biofilm cells while NEW-1 inhibited 0.3 log CFU/cm² only. Biofilms formation and antagonistic effect of NEW could be visualized by epifluorescence and scanning electron microscopy, revealing significant biofilms structure. The disinfectant effect of NEW may be attributed to the combined antimicrobial effect of available chlorine and high ORP exhibited by its oxidizing compounds. NEW does not promote metal equipment corrosion due to its neutral pH, and is also environmentally friendly.     

Biofilm formation of meat-borne Salmonella enterica and inhibition by the cell-free supernatant from Pseudomonas aeruginosa

Using polystyrene surfaces for attachment, we evaluated biofilm formation and cell surface hydrophobicities of 17 Salmonella enterica strains (belonging to 9 different serovars), and further assessed the effect of cell-free culture supernatants (CFS) from Pseudomonas aeruginosa (containing acylated homoserine lactones (AHLs)), on the growth and biofilm development of S. enterica. The results indicated that most of the 17 strains readily formed biofilms on polystyrene surfaces, but the development of biofilms were significantly influenced by serovars and incubation conditions. Strains of S1 (S. London), S2 (S. London), S9 (S. Indiana) and S16 (S. Typhimurium) produced greater biofilms regardless of the tested conditions compared with other strains, with maximum biofilm production of 3.08, 2.47, 2.21 and 3.39 (crystal violet assay), respectively. Significant differences in cell hydrophobicity were observed between strains, S16 (S. Typhimurium) showed highest hydrophobicity values (>56%) compared with the other strains (<46%). A significant positive correlation was observed between cell surface hydrophobicity and the capacity of individual strains to form biofilms. The presence of 30% and 60% CFS from P. aeruginosa (containing AHLs identified by thin-layer chromatography), significantly decreased the growth rates of S. enterica (S1, S9 and S16) during the exponential phase, but not during the stationary phase, and significantly inhibited biofilm development of S. enterica strains (S1, S9 and S16) when incubated both in tryptic soytone broth (TSB) and meat thawing loss broth (MTLB), reaching maximum inhibition percentage of 70.7 and 93.3, respectively. Our findings are therefore important for developing innovative control strategies of Salmonella biofilms.     

Synergetic antibacterial efficacy of cold nitrogen plasma and clove oil against Escherichia coli O157:H7 biofilms on lettuce

This study aims to evaluate the antimicrobial effects of cold nitrogen plasma (CNP) and clove oil against Escherichia coli O157:H7 (E. coli O157:H7) biofilm on lettuce. Both clove oil (1 mg/mL, 2 mg/mL and 4 mg/mL) and CNP (400–600 W) displayed significant eradication effect on E. coli O157:H7 biofilms in vitro (p < 0.001). Subsequently, the antibiofilm effect of combined treatment was studied as well. Compared with the respective treatment, combined treatment exhibited remarkable synergistic effect on eradicating E. coli O157:H7 biofilms. The confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) had also visually testified that the antibacterial effects of clove oil on E. coli O157:H7 biofilms (in vitro and on lettuce) were enhanced by CNP at 400 W for short treatment duration. The results of sensory evaluation indicated that combined treatment has mild negative effect on lettuce quality. Moreover, the synergetic antibacterial mechanism of clove oil and CNP against E. coli O157:H7 was concluded as that they could damage the bacterial cell wall and the outer membrane, leading to leakage of cellular components, such as nucleic acid and ATP.     

Effects of phenyllactic acid as sanitizing agent for inactivation of Listeria monocytogenes biofilms

3-Phenyllactic acid (PLA) has been reported as an antimicrobial compound with broad-spectrum activity, and it can be produced by food-grade microorganisms, including a wide range of lactic acid bacteria species. In this study, the efficacy of PLA to inactivate Listeria monocytogenes planktonic cells and biofilms was determined and compared with the killing effects of lactic acid (LA), and levulinic acid (LVA) with sodium dodecyl sulfate (SDS). L. monocytogenes biofilms of different maturities, i.e., 37 °C for 3 and 7 d and 15 °C for 4 and 7 d, were produced on 24-well flat-bottom polystyrene plates and treated with PLA (0.25%–3%), LA (1% and 3%), and 3% LVA plus 2% SDS for 5, 10, 30, 60 min, respectively. The results of pure culture assays revealed that 1% PLA reduced the population of L. monocytogenes by 7 log CFU/ml within 1 min. The biofilms assays revealed that L. monocytogenes biofilms could be inactivated to different degrees by the sanitizer treatments. The killing effect of PLA treatment was increased as exposure time and PLA concentrations were increased. The sanitizers of 3% PLA and 1% PLA effectively inactivated the early mature biofilm after a 5-min treatment, whereas 3% PLA was better than all the other sanitizers, including 1% PLA, 3% LA, 3% LVA and 2% SDS for inactivation of the late mature biofilm after a 5-min treatment. Confocal laser scanning microscopy analysis revealed that bacterial cell damages in the biofilm were enhanced as the PLA concentrations and exposure times were increased. These results suggested that PLA was effective in inactivating L. monocytogenes and its biofilm, even for the late mature biofilm.     

Nucleolytic enzymes from the marine bacterium Cobetia amphilecti KMM 296 with antibiofilm activity and biopreservative effect on meat products

Cultivation of Pseudomonas aeruginosa (P. aeruginosa), Bacillus subtilis (B.subtilis), Salmonella enterica (S. enterica), and Staphylococcus aureus (S. aureus) isolated from meat products together with the marine bacterium Cobetia amphilecti KMM 296 (Cobetia marina) resulted in inhibition of their cell growth and complete degradation of biofilms of P. aeruginosa and B. subtilis. The degradation patterns of their mature biofilms treated with Cobetia amphilecti (C. amphilecti) extracellular nucleolytic enzymes revealed that the highly active alkaline phosphatase CmAP could be a significant antibiofilm factor. Moreover, CmAP possessed strong dose-dependent inhibition effect on de novo biofilm formation by bacterial cells regardless of the species. The concentration of CmAP for exhibition of maximal effect on microbial growth and biofilms was 1.1 μg/ml with activity of 2.5 units/ml that diminished total aerobic mesophilic and lactic acid counts in sausage shells by 3.5 log units for 5 days and 2.5 log units for 6 days, respectively. The stabilized antifungal effect continued the same period of storage. The nuclease-like enzymes CmEEP and CmNUC were species-independent and largely degraded biofilms at lowered pH. The biopreservative effect on meat products at low concentration and psychrophilicity of CmAP can be used for enzymatic cleaning of surfaces in food industry.     

Removal of Listeria monocytogenes, Staphylococcus aureus and Escherichia coli O157:H7 biofilms on stainless steel using scallop shell powder

Biofilms on steel surfaces containing Listeria monocytogenes, Staphylococcus aureus and Escherichia coli O157:H7 continue to threaten dairy and meat processors. In this study, the ability of scallop shell powder (SSP) to remove biofilms formed by these three pathogens on stainless steel plates was examined. Whey powder solution (WPS) and bench wash water (BWW) provided by dairy and meat factories, respectively, were inoculated with L. monocytogenes, S. aureus or E. coli O157:H7 (9 log₁₀ CFU/ml). Stainless steel plates (10 cm²) were placed in the inoculated fluids and incubated at 20 °C at 48 h to form biofilms. After drying and washing in sterile water, the plates were treated with 0.0, 0.25, or 0.50% (w/v) SSP slurries for 1, 5, or 10 min and then quantitatively examined for the three pathogens. Both 0.25 and 0.50% SSP reduced L. monocytogenes on the plates by 4 log CFU/cm² with a 1 min exposure to 0.50% SSP decreasing S. aureus by 5 logs CFU/cm². After 1 min in 0.25 and 0.50% SSP, E. coli O157:H7 populations in WPS and BWW biofilms decreased 4 and 6 log CFU/cm² and 3 and 5 log CFU/cm², respectively. Increasing the concentration of SSP led to significantly increased efficacy against the tested pathogens (P < 0.05). In conclusion, this study showed that SSP slurries could significantly reduce the numbers of L. monocytogenes, S. aureus and E. coli O157:H7 in biofilms on stainless steel surfaces.     

Single- and mixed-species biofilm formation by Escherichia coli O157:H7 and Salmonella,and their sensitivity to levulinic acid plus sodium dodecyl sulfate

The development of single- and mixed-species biofilms formed by Escherichia coli O157:H7 and Salmonella was observed, and the antimicrobial effectiveness of levulinic acid (LVA) plus sodium dodecyl sulfate (SDS) on the cells in single- and dual-species biofilms was determined. Biofilm-forming ability of single- and mixed-species cultures was observed by crystal violet staining and their resistance to levulinic acid plus SDS was determined by enumeration. Fluorescent protein-labeled E. coli O157:H7 and Salmonella were constructed and the bacterial composition of the biofilms after treatment with levulinic acid plus SDS was visualized by confocal laser scanning microscopy (CLSM). E. coli O157:H7 and Salmonella were antagonistic to each other, being more sensitive to levulinic acid plus SDS in mixed-species biofilms. Images captured by CLSM revealed that E. coli O157:H7 and Salmonella were distributed evenly in the single- and dual-species biofilms, and confirmed that the combination of levulinic acid and SDS was effective in inactivating bacterial cells in biofilms. Results revealed that levulinic acid with SDS may be used as a potential biofilm control intervention.     

Control of milk pH reduces biofilm formation of Bacillus licheniformis and Lactobacillus paracasei on stainless steel

Microbial biofilms present in dairy farms may contaminate milk during milk harvest and transfer diseases from the environment to cows. In order to reduce biofilm formation with respect to the role of pH, a study involving the control of milk pH during long-term biofilm formation of Bacillus licheniformis NBRC 12195 and Lactobacillus paracasei subsp. paracasei NBRC 15889 on stainless steel coupons in different dilutions of skim milk (0.1%, 1.0% and 5.0%) was conducted. During long incubation at 30 °C, pH decreased due to bacterial development in unadjusted samples. In pH-adjusted samples, pH was kept at around 7.0 by the addition of sterile sodium hydroxide. Biofilms formed on stainless steel coupons were daily stained by 0.1% Crystal Violet solution and assessed by the evaluation of optical density. The bacterial count of the suspensions showed that the control of pH enhanced the growth of bacteria in free-floating form. In contrast, optical densities of biofilms formed in the pH-adjusted samples were significantly lower than in the pH-unadjusted samples in all of three skim milk dilutions. Comparison of maximum OD values of adhered cells at different nutrient levels also implicated that for both tested strains, thicker biofilms were formed in milk dilutions at higher nutrient levels. These results suggested that, control of milk pH and milk residue level could significantly reduce biofilm formation of the tested bacteria.     

Inactivation and induction of sublethal injury of Listeria monocytogenes in biofilm treated with various sanitizers

This study determined the effects of different sanitizers (one phenolic-based, one chlorine-based, two QACs-based and one levulinic acid and SDS-based) on Listeria monocytogenes biofilm. The induction of the sub-lethal injury state and the biofilm formation characteristics as a result of exposure to sanitizers were also evaluated. The results revealed that QACs-based and phenolic-based sanitizers most effectively reduced L. monocytogenes, resulting in a reduction of 3.7–6.9 log CFU/ml and 4.9–8.2 log CFU/ml after a 60-min treatment for 37°C- and 15°C-grown biofilms, respectively. An enhanced level of sanitizer resistance was observed in biofilms when they were multiply exposed to QACs-based and phenolic-based sanitizers, with a reduction of 0.7–3.5 log CFU/ml and 1.6–>9.3 log CFU/ml for 37°C- and 15°C-grown biofilms, respectively. As biofilm cells became less sensitive, especially to QACs-based sanitizers, an increase in the percentage of sublethally injured cells was observed to the levels dependent upon sanitizer concentration. Confocal laser scanning microscopy (CLSM) analysis revealed that biofilm cells experienced cell membrane damage when exposed to QACs-based and phenolic-based sanitizers, providing more protection to cells located inside the biofilm matrix. This study highlights the ongoing need for improvement in intervention methods to control L. monocytogenes in food processing plants.     

Production of biofilm by Listeria monocytogenes in different materials and temperatures

Listeria monocytogenes, considered as one of the most important foodborne pathogens, is easily found on surfaces, particularly in the form of a biofilm. Biofilms are aggregates of cells that facilitate the persistence of these pathogens in food processing environments conferring resistance to the processes of cleaning and may cause contamination of food during processing, thus, representing a danger to public health. Little is known about the dynamics of the formation and regulation of biofilm production in L. monocytogenes, but several authors reported that the luxS gene may be a precursor in this process. In addition, the product of the inlA gene is responsible for facilitating the entry of the microorganism into epithelial cells that express the receptor E-cadherin, also participates in surface attachment. Thus, 32 strains of L. monocytogenes isolated from different foods (milk and vegetables) and from food processing environments were analyzed for the presence of these genes and their ability to form biofilms on three different surfaces often used in the food industry and retail (polystyrene, glass and stainless steel) at different temperatures (4, 20 and 30 °C). All strains had the ilnA gene and 25 out of 32 strains (78.1%) were positive for the presence of the luxS gene, but all strains produced biofilm in at least one of the temperatures and materials tested. This suggests that genes in addition to luxS may participate in this process, but were not the decisive factors for biofilm formation. The bacteria adhered better to hydrophilic surfaces (stainless steel and glass) than to hydrophobic ones (polystyrene), since at 20 °C for 24 h, 30 (93.8%) and 26 (81.3%) produced biofilm in stainless steel and glass, respectively, and just 2 (6.2%) in polystyrene. The incubation time seemed to be an important factor in the process of biofilm formation, mainly at 35 °C for 48 h, because the results showed a decrease from 30 (93.8%) to 20 (62.5%) and from 27 (84.4%) to 12 (37.5%), on stainless steel and glass, respectively, although this was not significant (p = 0.3847). We conclude that L. monocytogenes is capable of forming biofilm on different surfaces independent of temperature, but the surface composition may be important factor for a faster development of biofilm.     

Antibiofilm activity of essential oils and plant extracts against Staphylococcus aureus and Escherichia coli biofilms

Bacterial biofilms pose health risks in clinical environments, food industry and drinking water systems. Here, we investigated in vitro antibiofilm activities of essential oils (EO) and plant extracts of peppermint (Mentha × piperita L.), coriander (Coriandrum sativum L.), and anise (Pimpinella anisum L.). Minimum inhibitory concentration assay (MIC) was carried out using two-fold serial dilution method and MTT assay against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria. Biofilm growth and development were assessed using crystal violet (CV) and XTT reduction assays. Antibacterial activity was observed for almost all plant extracts and all EOs against both bacterial strains with stronger activity against S. aureus. All EOs (at MIC value of 0.8 to 0.63 μl/ml) and 8 out of 14 plant extracts (at MIC value of 2–4 mg/ml) inhibited bacteria cell attachment of both bacteria. CV and XTT reduction assay for the plant extracts and EOs with inhibition of bacteria attachment by at least 50%, demonstrated that coriander EO had the highest antibiofilm activity against biofilm formed by both tested bacteria (S. aureus and E. coli) at lowest MIC value 0.8 μl/ml and 1.6 μl/ml, respectively, indicating further investigations due to the oil's high antibiofilm activity potential.     

Influence of temperature and surface kind on biofilm formation by Staphylococcus aureus from food-contact surfaces and sensitivity to sanitizers

This study aimed to assess the adhesion, detachment kinetic and biofilm formation of Staphylococcus aureus isolates from food services surfaces on stainless steel and polypropylene surfaces when cultivated in a vegetable-based broth at 7 and 28 °C, and the efficacy of peracetic acid (30 mg/L) and sodium hypochlorite (250 mg/L) in removing the bacterial cells from the matrix of the preformed biofilm. The isolates adhered over 4 Log cfu/cm² regardless the surface kind and incubation temperature. Cell detachment was around 3 Log cfu/cm² over the first six contacts with agar characterizing a high persistence of cells on the tested surfaces. Number of cells (5-7 Log cfu/cm²) needed for biofilm formation was noted at all experimental systems already after 3 days of incubation. A range of 2.0-3.3 and 1.5 to 2.1 Log cfu/cm² was observed in the reduction of cells in biofilm matrix caused by peracetic acid and sodium hypochlorite, respectively. The isolates of S. aureus revealed high capability to adhere and form biofilm on the tested surfaces in both assayed incubation temperature.