Assessing biofilm formation by Salmonella enterica serovar Typhimurium on abiotic substrata in the presence of quorum sensing signals produced by Hafnia alvei

This study aimed to evaluate the possible impact of N-acyl-homoserine lactones (AHLs) produced by Hafnia alvei on the biofilm-forming ability of Salmonella enterica ser. Typhimurium. To do this, S. Typhimurium was left to form biofilms on two abiotic substrata, stainless steel (SS) and polystyrene (PS), through their static incubation for up to 48 h, at 20 °C, in laboratory growth media, also containing crude or ethyl acetate extracted cell-free culture supernatant of either the AHL-producing H. alvei 718 strain or its AHL-lacking isogenic mutant. Apart from the presence of signals, two nutritional conditions, a rich and a poor one, were also evaluated in those experiments involving the ethyl acetate extracted supernatants. The presence of AHLs in all media was tested using Agrobacterium tumefaciens biosensor-based bioassay, while the quantities of biofilms formed were in parallel estimated through three different quantification methods, namely agar plating, conductance, and absorbance measurements. Results revealed a significant correlation in biofilm formation among both tested surfaces, with the conductance detection times to be also inversely well correlated to the biofilm counts on SS. Interestingly, S. Typhimurium in general presented a higher capability to form biofilms under the nutrient poor compared to rich conditions. However, all hereby employed biofilm setup and quantification methods did not reveal any significant influence of H. alvei AHLs on Salmonella biofilm-forming behavior. In agreement, further experiments conducted using a synthetic AHL (i.e.3OC6-HSL), simulating a major such signal produced by H. alvei, did not show any effect. To sum up, these results expand our knowledge on the potential role of interspecies signaling on biofilm formation by S. Typhimurium.     

Modelling survival behaviour of Salmonella enterica ser. Enteritidis,Typhimurium and Tennessee on table eggs during storage at different temperatures

Quantitative risk assessment studies on the health risk of Salmonella due to consumption of contaminated table eggs are based on the assumption that Salmonella is inside the egg and that the pathogen belongs to serovar Enteritidis. However different serovars of Salmonella may contaminate the surface of table eggs and spread to other foods at consumer's kitchen due to improper food handling. In the present study the survival behaviour of one strain each from Salmonella enterica serovars Enteritidis, Typhimurium and Tennessee on table egg surface during storage at 4, 8, and 20 °C have been described. Besides, in those cases observed data were subjected for modelling; linear, log-linear tail and Weibull models were compared in terms of model fitting and model performance. Overall, in most cases, inactivation kinetics presented a linear trend on Salmonella behaviour so that Weibull and linear models adequately described observed data. Regarding log-linear tail models, though they presented a better fitting, their adequacy could not be assessed given the lack of data in the tail region. Regarding storage temperatures, 4 °C was predicted to be the most inhibitory temperature for table eggs externally contaminated by a strain of S. enterica serovar Enteritidis. After 28 days of storage, a reduction of 4 log₁₀ cfu/g of eggshell on the S. enterica ser. Enteritidis load was registered at 4 °C. S. enterica ser. Typhimurium and Tennessee showed higher survival rates at all tested temperatures. The results highlighted the importance of keeping constant the storage temperature of table eggs in order to reduce the risk of S. enterica ser. Enteritidis contaminating the surface of table eggs. However this temperature might not be the optimal one in view of S. enterica serovars other than Enteritidis.     

Fiber optic and light scattering sensors: Complimentary approaches to rapid detection of Salmonella enterica in food samples

Salmonella-related foodborne infections present a major public health problem worldwide despite more stringent regulations. Salmonella enterica serovars Enteritidis and Typhimurium are the two most frequent causes of poultry related outbreaks; therefore, their rapid and accurate detection would improve Salmonella control at the farm, processing plant, and at retail. In this study, we investigated if a fiber optic immunosensor and light scattering sensor, BARDOT (bacterial rapid detection using optical scattering technology) could facilitate the detection of these two serovars in naturally contaminated poultry (n = 50). The fiber optic sensor with a detection limit of 10³ CFU/ml identified S. enterica in selective enrichment broth in less than 12 h. The colonies (1.0 ± 0.2 mm) produced by plating the enriched samples on selective XLT4 agar for 13–15 h were scanned using BARDOT and S. enterica was identified after matching individual colony scatter patterns to the scatter image library with a sample-to-answer time of about 24 h. Both sensors identified 4 positive samples (8%), which corresponded to the results of the USDA-FSIS protocol, PCR, and lateral flow immunoassays. The colony scatter patterns identified all natural isolates as S. Enteritidis, which was further verified by serovar-specific PCR. The sensors used individually or in combination demonstrate potential for accurate and rapid detection of S. enterica in poultry.     

Prevalence,genetic characterization and antimicrobial resistance of Salmonella isolated from fresh pork sausages in Porto Alegre,Brazil

A total of 336 samples of fresh pork sausage randomly obtained from supermarkets and butcher shops in Porto Alegre, Brazil, were examined for the presence of Salmonella serovars. Salmonella enterica was detected in 82 (24.4%) of the samples, with a most probable number count ranging from 0.03 MPN g^?1 to 460 MPN g^?1. Strains belonging to the most isolated S. enterica serovars (Brandenburg, Panama, Derby and Typhimurium) were further characterized by XbaI-macrorestriction, resulting in a total of 17 profiles. Resistance to tetracycline was the most prevalent among the Salmonella isolates. S. panama and S. typhimurium presented the greatest number of resistance phenotypes.     

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.     

The sanitizing action of essential oil-based solutions against Salmonella enterica serotype Enteritidis S64 biofilm formation on AISI 304 stainless steel

Bacterial adhesion and biofilm formation by Salmonella enterica serovar Enteritidis is a serious concern in the food processing industry; organism persistence in biofilms represents a continual source of contamination. Due to unsuccessful disinfection processes and emerging resistance, conventional control methods are rapidly becoming ineffective, necessitating the development of new control strategies. The following study evaluated the anti-biofilm effect of disinfectant solutions formulated with essential oils (EOs) of peppermint (Mentha piperita) and lemongrass (Cymbopogon citratus) against biofilm formation by S. enterica serotype Enteritidis S64 on stainless steel surface AISI 304 (#4) after 10, 20 and 40 min of contact. A minimum inhibitory concentration (MIC) of 7.8 μL/mL was found for both EOs and disinfectant solutions were formulated based on these MIC values. Ten minutes of sanitizing solution contact significantly reduced (p ≤ 0.05) adhered bacterial populations for both EOs tested. After 20 and 40 min of treatment, cell counts were not detected. Thus, M. piperita and C. citratus EOs can be considered convenient, quality alternatives to the application of conventional sanitizing agents in the food industry; further, use of these EOs addresses the increasing consumer demand for natural products.     

A comparison of saturated steam and superheated steam for inactivation of Escherichia coli O157:H7, Salmonella Typhimurium,and Listeria monocytogenes biofilms on polyvinyl chloride and stainless steel

This study was performed to compare the effectiveness of saturated steam (SS) and superheated steam (SHS) in the inactivation of Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes biofilms on polyvinyl chloride (PVC) and stainless steel. Biofilms were formed on PVC and stainless steel coupons by using a mixture of three strains each of three foodborne pathogens at 25 °C. After biofilm development, PVC and stainless steel coupons were treated with SS at 100 °C and SHS at 125, 150, 175, and 200 °C for 5, 10, 20, and 30 s on both sides. The viable cell numbers of biofilms were significantly (p < 0.05) reduced as SHS temperature and exposure time increased. For all biofilm cells, SHS treatment resulted in an additional log reduction compared to SS treatments. After exposure to 200 °C steam for 30 s or 10 s on PVC or stainless steel, respectively, the numbers of biofilm cells were reduced to below the detection limit (1.48 log). This study demonstrated that SHS treatment effectively reduced populations of biofilm cells and reduced disinfection time compared to SS treatments and further evaluated its potential as an excellent intervention for controlling microbial biofilms and enhancing safety in the food processing industry.     

Sextuplex PCR combined with immunomagnetic separation and PMA treatment for rapid detection and specific identification of viable Salmonella spp., Salmonella enterica serovars Paratyphi B,Salmonella Typhimurium,and Salmonella Enteritidis in raw meat

Salmonella is one of the most common pathogens that cause foodborne diseases in humans, resulting in high medical and economical costs worldwide. The aim of this study was to develop a rapid and accurate approach for simultaneous detection of viable Salmonella spp. in raw meat, including Salmonella enterica serovars Paratyphi B, S. Typhimurium, and S. Enteritidis. To reduce detection time and improve sensitivity, immunomagnetic separation (IMS) was used as a pre-concentration and separation method. In addition, false positive and false negative results were removed by combining propidium monoazide (PMA) treatment with internal amplification control. Results showed that the detection limit of IMS-PMA combined with sextuplex polymerase chain reaction (PCR) assay reached as low as 10¹ CFU/mL in pure culture and 10² CFU/g in spiked raw meat (ground pork and ground beef), and the total assay time took less than 6 h. Thus, the novel IMS-PMA-mPCR assay developed in this study holds promise for routine screening of viable Salmonella serovars in meat and other samples.     

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.     

Detection and CRISPR subtyping of Salmonella spp. isolated from whole raw chickens in Yangzhou from China

This study was undertaken to acquire data on Salmonella contamination of whole raw chickens, eggs, and vegetables available to consumers in Yangzhou city, China, between April 2011 and March 2012. In total, 240 chicken carcasses were tested, and the overall contamination rate for Salmonella was 33.8%. While the prevalence of Salmonella in 100 eggs and 155 vegetable samples was 7.0% and 3.2%, respectively. The 84 isolated strains were identified in 19 different serotypes with Salmonella enterica serovar Indiana (S. Indiana) (25.0%), S. Typhimurium (21.4%) and S. Enteritidis (17.9%) as the predominant serovars. Moreover, the median load of the contaminated chicken samples reached 6.4 MPN/100 g with 3.6 MPN/100 g as the 25th percentile and 15.0 MPN/100 g as the 75th percentile. Chicken carcasses collected in October had not only the highest prevalence of Salmonella (70%), but also the highest median load (33 MPN/100 g) and 75th percentile load (460 MPN/100 g), while the lowest prevalence (10%) was in April. The clustered regularly interspaced short palindromic repeats (CRISPR) subtyping method was then used to identify serotypes of Salmonella and distinguish strains from the same Salmonella serotypes. We found that 85.7% of strains were distributed in 11 serotypes speculated by CRISPR typing, which corresponded to the identified serotypes by O and H antiserum. The speculated serotypes of 7.1% of the strains by CRISPR typing are very close to the identified ones, as they belong to the same O group with a small difference in the O or H antigen. All of the above findings implied that CRISPR typing could be applied to serotyping of Salmonella. In addition, CRISPR typing method could be used to subtype different strains from the same serotype, specifically S. Hadar.     

Biofilm formation by Salmonella Typhimurium and Staphylococcus aureus on stainless steel under either mono- or dual-species multi-strain conditions and resistance of sessile communities to sub-lethal chemical disinfection

Intercellular interactions encountered within and between different bacterial species are believed to play key roles in both biofilm formation and antimicrobial resistance. In this study, Salmonella Typhimurium and Staphylococcus aureus (3 strains per species) were left to form biofilms on stainless steel coupons incubated at 20 °C for 144 h (i.e. 6 days), in periodically renewable growth medium, under either mono- or dual-species conditions. Subsequently, the developed sessile communities were exposed for 6 min to sub-lethal concentrations of: (i) benzalkonium chloride (BC, 50 ppm), (ii) sodium hypochlorite (NaClO, 10 ppm), or (iii) peroxyacetic acid (PAA, 10 ppm). The dominance of each strain in the mono- and dual-species biofilm communities, both before and after disinfection, was monitored by pulsed field gel electrophoresis (PFGE). Results revealed that dual-species conditions led to a significant (ca. 10-fold) reduction in the number of sessile cells for both species, compared to mono-species ones, with interspecies interactions however found to not exert any significant effect on the disinfection resistance of each species as a whole. However, PFGE analysis revealed that the different strains here employed behaved differently with regard to biofilm formation and disinfection resistance, with this effect to be also strongly dependent on the culture conditions (mono-/dual-species) and the disinfectant applied. Such results expand our knowledge on multi-species biofilms formed by foodborne pathogenic bacteria and could hopefully be helpful in our efforts to develop effective elimination strategies and thus improve food safety.     

The specific antibacterial effect of the Salvia oil nanoliposomes against Staphylococcus aureus biofilms on milk container

The Staphylococcus aureus (S. aureus) biofilm on container is the main source of microbial contamination in milk. In this study, the nanoliposomes encapsulating Salvia oil (SO) has been prepared. Based on the damage effect of pore-forming toxin on cell membrane, α-toxin secreted from S. aureus has been used to trigger the release of SO from nanoliposomes to achieve antibacterial effect on S. aureus biofilm on milk container. Firstly, the minimum biofilm eradication concentration (MBEC) and biofilms time-dependent killing of SO were tested. The results showed that the MBEC of SO against S. aureus biofilms was 0.2% and SO effectively eradicated the biofilms after treating for 4 h. Subsequently, SO was encapsulated into nanoliposomes in order to increase its stability. The particle size, poly dispersity index (PDI), zeta potential, pH, turbidity, and entrapment efficiency of SO nanoliposomes were analyzed systematically. Gas chromatography–mass spectrometry (GC–MS) has been utilized to observe the controlled release of SO form nanoliposomes incubated with S. aureus. The scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) images have also visually showed that SO nanoliposomes have high anti-biofilm activity against S. aureus biofilms on milk container.     

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.     

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.     

Thermal death times of acid-habituated Escherichia coli and Salmonella enterica in selected fruit beverages

The study established the decimal reduction times at 60 and 73 °C (D₆₀ and D₇₃) of each of acid-adapted cocktails of Escherichia coli (NRRL 3704, ATCC 8739, ATCC 92522) and Salmonella enterica serovars Typhimurium (NRRL B-4420), Typhi (NRRL B-573), and Enteritidis (Biotech 1963) in some fruit beverages. Tested beverages included apple and orange juices, which are most commonly reported vectors of diseases; and tropical fruit beverages such as mango, guava, and soursop nectars, which are not frequently used as suspending media in thermal inactivation studies. The fruit beverages had pH of 3.30–4.73, titratable acidities of 0.15–0.64% organic acid, and soluble solids of 11.13–14.33 °Brix. At 60 °C, E. coli and Salmonella were 1.26–3.13 and 1.21–2.33 folds, respectively, more resistant to heating than at 73 °C. At 60 °C, E. coli and S. enterica had D₆₀ value ranges of 5.90 s (orange juice) to 12.42 s (guava nectar) and 7.50 s (orange juice) to 11.46 s (soursop nectar), respectively. At 73 °C, E. coli had D₇₃ values ranging from 3.56 s (apple juice) to 5.82 s (soursop nectar), while those of S. enterica ranged from 3.59 s (guava nectar) to 9.74 s (soursop nectar). The variations in the observed heat resistance in both heating temperatures were attributed to the differences in the physicochemical properties of the suspending fruit beverages. The results obtained in this work contribute to further understanding the behaviors of these pertinent pathogens in heat-treated fruit beverages. These data also provide baseline information for the establishment of heat pasteurization process schedules for better control of fruit juice product safety.     

Biofilm forming Salmonella strains exhibit enhanced thermal resistance in wheat flour

The biofilm lifestyle of bacteria confers a remarkably increased tolerance to antimicrobial interventions and environmental stresses, however little is known about influence of biofilms on thermal resistance of Salmonella in low-moisture foods. This study was aimed to assess the correlation between the ability of biofilm formation of Salmonella Enteritidis (S. Enteritidis) strains and their capacity to survive desiccation and thermal treatment in wheat flour as a model for low-moisture food. The production of the biofilm in S. Enteritidis strains was analyzed qualitatively and quantitatively using calcoflour fluorescence, congo-red binding, pellicle formation and microtiter-plate test. Subsequently, three biofilm-forming and four non-forming S. Enteritidis strains were selected. Survival after desiccation was evaluated by population counts before and after equilibration for 4–5 days at 45% RH. Thermal resistance (D_{80 °C, 0.45 aw}) of S. Enteritidis in wheat flour was evaluated by fitting the thermal inactivation kinetic data with the first order kinetics model. The biofilm forming ability was not associated with resistance to desiccation. However, thermal resistance (D_{80 °C, 0.45 aw}) and pre-formed biofilm amount (OD_492 nm) showed a linear correlation (Spearmen correlation ρ = 0.8, p < 0.05), indicating more biofilm production confers more thermal resistance. Average thermal resistance (D_{80 °C, 0.45 aw}) was significantly (p < 0.05) higher among biofilm formers (14.1 ± 0.6 min) when compared with non-formers (6.0 ± 0.2 min). This study shows that the amount of biofilm produced by Salmonella on congo red-calcofluor media is linearly correlated with the thermal resistance of Salmonella in wheat flour. The findings reinforce the necessity of appropriate management in sanitation and biofilm removal in plants that process low-moisture foods.     

Development of a multiplexed PCR assay combined with propidium monoazide treatment for rapid and accurate detection and identification of three viable Salmonella enterica serovars

Salmonella is the leading pathogenic bacteria in food and contaminated water. The aim of this study was to develop a rapid and reliable technique for simultaneous detection of the main three serotypes (Salmonella enterica serovars Typhimurium, Paratyphi B and Typhi) of Salmonella. Primers were designed to amplify the genes specific to each of these three serotypes for simultaneous detection using polymerase chain reaction (PCR). To ensure the detection of only viable cells, propidium monoazide (PMA) was applied to selectively suppress the DNA signal from dead cells. Results showed that the PMA-multiplexed PCR (PMA-mPCR) assay always gave negative results for heat-killed Salmonella at concentrations up to 1 × 10⁶ CFU/ml in pure culture or 1 × 10⁶ CFU/g in spiked food products (tomato, chicken, beef and ham). Results showed that the detection limits of the PMA-mPCR assay were approximately 10² CFU/ml (4.3 × 10² CFU/ml for S. Typhimurium, 3.7 × 10² CFU/ml for S. Paratyphi B, 7.2 × 10² CFU/ml for S. Typhi) in pure culture and 10³ CFU/g (4.3 × 10³ CFU/g for S. Typhimurium, 3.7 × 10³ CFU/g for S. Paratyphi B, 7.2 × 10³ CFU/g for S. Typhi) in food produce. These results demonstrated that the PMA-mPCR assay can simultaneously detect and identify viable S. Typhimurium, Paratyphi B and Typhi in a short period of time, even in food produce.     

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.     

Biofilm formation of Campylobacter strains isolated from raw chickens and its reduction with DNase I treatment

Campylobacter is a well-known bacterial agent that causes foodborne gastroenteritis. Its biofilm-forming ability is known to be important for its survival in harsh conditions. Poultry is a main reservoir of Campylobacter. In this study, we evaluated the biofilm-forming ability, motility, and antibiotic susceptibility of Campylobacter strains isolated from commercially purchased chickens of various sources in South Korea. From 37 (30%) out of 124 chickens, a total of 78 Campylobacter isolates were obtained, and the ability of these strains to form biofilms was studied on polystyrene surfaces. We identified seven biofilm-forming strains of Campylobacter jejuni and Campylobacter coli, respectively, out of 60 C. jejuni and 18 C. coli strains. Our study demonstrated that motility is inconsistent with biofilm-forming ability, suggesting that motility is not a single factor affecting biofilm formation of Campylobacter. Moreover, there was no clear relationship between antibiotic resistance and biofilm-forming ability. DNase I treatment significantly inhibited the biofilm formation or degraded the mature biofilms of 3 C. coli and 1 C. jejuni biofilm-forming strains (p < 0.05). It suggests that extracellular DNA plays a significant role in the biofilm formation of these strains. Collectively, our study demonstrated that biofilm-forming ability is not strongly correlated with motility and antibiotic resistance in Campylobacter and that extracellular DNA is required for biofilm formation of the isolated Campylobacter strains. It also suggests that DNase I is a potential method for the control of Campylobacter biofilms.     

Adherence kinetics, resistance to benzalkonium chloride and microscopic analysis of mixed biofilms formed by Listeria monocytogenes and Pseudomonas putida

Comparison between the resistance to BAC and the microscopic structure between mixed-species biofilms formed by different strains of Listeria monocytogenes and Pseudomonas putida CECT 845 under different scenarios and that obtained by the corresponding monospecies L. monocytogenes biofilm was carried out. The association of P. putida with L. monocytogenes quickens biofilm formation and increases significantly (p < 0.05) the BAC-resistance of the biofilm after 4 days of incubation at 25 °C respecting to that formed by monospecies biofilms. According with the adherence profiles of P. putida, two different patterns of association between both species (A and B) were identified, being type A pattern found in the mixed biofilms much more resistant to BAC. After 11 days of incubation, a destructuration of mixed biofilms occurred in all experimental assays, being in 2 out of 5 experimental cases (4032 and BAC-adapted 5873 on polypropylene) accompanied by a sharp decrease in the number of adhered cells. Microscopic analyses demonstrated that complex three-dimensional microscopic structure showed the highest resistance to BAC (4032-SS). Obtained results clearly highlight that to improve disinfection protocols for assuring food safety, it is necessary to mimick those bacterial association that occur in nature.