Effect of chemical modifications of tannins on their antibiofilm effect against Gram-negative and Gram-positive bacteria

Background Tannins have demonstrated antibacterial and antibiofilm activity, but the mechanisms of action are not completely elucidated. We are interested in understanding how to modulate the antibiofilm activity of tannins and in delineating the relationship between chemical determinants and antibiofilm activity. Materials and methods the effect of five different naturally acquired tannins and their chemical derivatives on biofilm formation and planktonic growth of Salmonella Typhimurium, Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus was determined in the Calgary biofilm device. Results most of the unmodified tannins exhibited specific antibiofilm activity against the assayed bacteria. The chemical modifications were found to alter the antibiofilm activity level and spectrum of the tannins, with the positive charge introducing C3NMe3Cl-0.5 derivatization shifting the anti-biofilm spectrum towards Gram-negative bacteria and C3NMe3Cl-0.1 and the acidifying CH3COOH derivatization shifting the spectrum towards Gram-positive bacteria. Also, the quantity of phenolic-OH groups per molecule has a weak impact on the anti-biofilm activity of the tannins. Conclusions we were able to modulate the antibiofilm activity of several tannins by specific chemical modifications, providing a first approach for fine tuning of their activity and spectrum

Occurence and characterisation of biofilms in drinking water systems of broiler houses

Background: Water quality in the drinking water system (DWS) plays an important role in the general health and performance of broiler chickens. Conditions in the DWS of broilers are ideal for microbial biofilm formation. Since pathogens might reside within these biofilms, they serve as potential source of waterborne transmission of pathogens to livestock and humans. Knowledge about the presence, importance and composition of biofilms in the DWS of broilers is largely missing. In this study, we therefore aim to monitor the occurrence, and chemically and microbiologically characterise biofilms in the DWS of five broiler farms. Results: The bacterial load after disinfection in DWSs was assessed by sampling with a flocked swab followed by enumerations of total aerobic flora (TAC) and Pseudomonas spp. The dominant flora was identified and their biofilm-forming capacity was evaluated. Also, proteins, carbohydrates and uronic acids were quantified to analyse the presence of extracellular polymeric substances of biofilms. Despite disinfection of the water and the DWS, average TAC was 6.031.53 log CFU/20cm(2). Enumerations for Pseudomonas spp. were on average 0.88 log CFU/20cm(2) lower. The most identified dominant species from TAC were Stenotrophomonas maltophilia, Pseudomonas geniculata and Pseudomonas aeruginosa. However at species level, most of the identified microorganisms were farm specific. Almost all the isolates belonging to the three most abundant species were strong biofilm producers. Overall, 92% of all tested microorganisms were able to form biofilm under lab conditions. Furthermore, 63% of the DWS surfaces appeared to be contaminated with microorganisms combined with at least one of the analysed chemical components, which is indicative for the presence of biofilm. Conclusions: Stenotrophomonas maltophilia, Pseudomonas geniculata and Pseudomonas aeruginosa are considered as opportunistic pathogens and could consequently be a potential risk for animal health. Additionally, the biofilm-forming capacity of these organisms could promote attachment of other pathogens such as Campylobacter spp. and Salmonella spp

Effect of chemical modifications of tannins on their antimicrobial and antibiofilm effect against Gram-negative and Gram-positive bacteria

BackgroundTannins have demonstrated antibacterial and antibiofilm activity, but there are still unknown aspects on how the chemical properties of tannins affect their biological properties. We are interested in understanding how to modulate the antibiofilm activity of tannins and in delineating the relationship between chemical determinants and antibiofilm activity.Materials and methodsThe effect of five different naturally acquired tannins and their chemical derivatives on biofilm formation and planktonic growth of Salmonella Typhimurium, Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus was determined in the Calgary biofilm device.ResultsMost of the unmodified tannins exhibited specific antibiofilm activity against the assayed bacteria. The chemical modifications were found to alter the antibiofilm activity level and spectrum of the tannins. A positive charge introduced by derivatization with higher amounts of ammonium groups shifted the anti-biofilm spectrum toward Gram-negative bacteria, and derivatization with lower amounts of ammonium groups and acidifying derivatization shifted the spectrum toward Gram-positive bacteria. Furthermore, the quantity of phenolic OH-groups per molecule was found to have a weak impact on the anti-biofilm activity of the tannins.ConclusionWe were able to modulate the antibiofilm activity of several tannins by specific chemical modifications, providing a first approach for fine tuning of their activity and antibacterial spectrum

The influence of biofilms in the food processing environment on the spoilage of ready-to-heat sauces

Food spoilage is the microbial, chemical or physical change of a product causing it to become undesirable or unacceptable for human consumption. It is generally considered that microbial spoilage is the major cause of processed food spoilage. Microbial food spoilage is caused by a sequence of events: spoilage organisms gain access to the product; they survive and adapt to the product; and they grow to sufficiently high cell counts to cause a change in the food product. Biofilms are consortia of bacteria adhering to a surface and enveloped in a self-produced extracellular matrix. By producing biofilms, bacteria have a higher tolerance against cleaning and disinfection protocols compared to their planktonic (free-living) counterpart and may thus survive in the food processing environment. Therefore, introduction to the food product from these biofilms is possible and the presence of biofilms is often considered as a risk for the safety and quality of the food products. Thus, the goal of this PhD research was to examine the influence of biofilms in the food processing equipment on the spoilage of food products with ready-to-heat (RTH) sauces as a case study. For this, the following approach was used: we (i) isolated the microorganisms of prematurely spoiled RTH-sauces and the food processing environment of these products, (ii) determined the biofilm-forming potential of these isolates, (iii) ascertained the biofilm tolerance against conventional cleaning and disinfection (C&D) of a selection of these isolates in a reactor model which could simulate in situ C&D conditions, (iv) checked the effect of the interactions between food spoilage bacteria, with either low or high biofilm-forming ability, and the isolates from the food processing environment on the survival of the food spoilage bacteria in the food processing equipment to (v) finally evaluate both a preventive and curative biofilm removal strategy. In a first step, the spoilage bacteria of RTH sauces, which showed signs of premature spoilage, were isolated and identified. Three main spoilage organisms were found with different spoilage profiles. Bacilli were most often isolated and appeared to cause mild acidification and liquefaction in the RTH-sauces. Lactic acid bacteria were the second most isolated and were associated with severe acidification of the product. Finally, although only infrequently isolated, yeasts caused gas formation in the food product. The presence of bacilli in the food product might be explained by their ability to form spores which enhances their heat tolerance and might allow them to survive the heat treatment during processing of the sauces. All other spoilage bacteria, however, were not able to survive this heat treatment, indicating a secondary contamination of the sauces during filling. Moreover, the presence of biofilms in the filling equipment of these sauces was shown in a previous study, pointing to the presence of biofilms as a risk for the contamination of the RTH-sauces. However, the spoilage bacteria, isolated from the RTH-sauces, were not found among the dominant bacteria, found in biofilms in the filling equipment, indicating that even at low abundancies in the food processing environment, the spoilage bacteria might pose a risk for contamination and consequent spoilage of the RTH sauces. Subsequently, the biofilm-forming ability of both kinds of isolates was determined in vitro. The majority of the food spoilage isolates showed some degree of biofilm formation, except a number of lactic acid bacteria. The biofilm forming ability of the isolates from the filling equipment of RTH-sauces were compared to isolates from processing equipment of other food sectors, in an attempt to find similarities between the different food sectors. However, few similarities were found among the strong biofilm producers of the different companies, except for Pseudomonas spp. and Stenotrophomonas maltophilia, which were found among the strong biofilm producers in every company except one. To determine the biofilm tolerance against conventional cleaning and disinfection, first a laboratory method was designed to study the (organic fouling and) biofilm removal from stainless steel which could simulate in situ cleaning and disinfection conditions. This biofilm reactor model was used in the next part of the research to characterize the tolerance against cleaning and disinfection of the spoilage bacteria, isolated from the RTH-sauces, and the food processing bacteria, isolated from biofilms in the filling equipment. The spoilage bacteria were able to form biofilms on stainless steel surfaces and survive cleaning and disinfection, albeit at low initial cell counts before cleaning and disinfection and/or low tolerance against cleaning and disinfection which coincided with their (hypothesized) low abundancy in situ. In contrast, biofilms of isolates from the food processing environment showed both high initial cell counts in biofilm and high residual cell counts after cleaning and disinfection. The presence of these strong biofilm producing bacteria in the food processing environment might influence the attachment and survival of the relatively low biofilm producing food spoilage strains, which was investigated in a later step of the research. In a final step of this part, the biofilm tolerance of the strongest biofilm producers of several food companies was compared. A low efficacy of the disinfectants, without prior cleaning, was observed. However, when a cleaning step was incorporated, the efficacy of the total cleaning and disinfection protocol increased, mainly due to a high biofilm removal during cleaning. Nonetheless, differences in regrowth of the residual biofilm after (cleaning and) disinfection were observed between the different bacteria. For some bacteria the number of cells after regrowth was comparable to or higher than the initial biofilm cell count, independent of the incorporation of a cleaning step or the cleaning and disinfection products used. For one strain, the use of chlorinated products induced a higher regrowth compared to the other cleaning agents and disinfectants. For the remaining bacteria, the incorporation of a cleaning step significantly inhibited the regrowth to cell counts below the initial biofilm cell count before cleaning and disinfection, independent of the cleaning and disinfection products used. These results indicated that (i) cleaning and disinfection should be optimized against the specific bacteria isolated from the food processing environment and (ii) that further optimization of the cleaning and disinfection protocols for biofilm removal and control is still needed. Given the low biofilm forming ability and/or tolerance against cleaning and disinfection of the food spoilage bacteria, we hypothesized that the strong biofilm producing bacteria present in the food processing environment might increase the survival of the food spoilage bacteria. Therefore, in a next step, the isolates from biofilms in the filling equipment of the RTH-sauces were pairwise cocultured with the spoilage bacteria, isolated from the RTH-sauces, to determine their interactions. For most combinations, a decrease in fitness of the spoilage bacteria, with a decrease of the total biomass and EPS production, was observed. The only exception were the combinations of food spoilage bacteria with S. maltophilia, which instead showed a positive complementary effect for all food spoilage bacteria. Therefore, these combinations were further characterized for their social interactions and tolerance against cleaning and disinfection in the biofilm reactor model, indicating a dual role of S. maltophilia on the attachment and survival of different spoilage bacteria. The fitness and tolerance against cleaning and disinfection of bacilli decreased when co-cultured with S. maltophilia, as opposed to the fitness and tolerance of lactic acid bacteria, which increased when cocultured. The presence of S. maltophilia in the food processing environment might thus both prevent or increase the risk of transmission of spoilage bacteria to the food product, dependent on the specific spoiler. Therefore, in the final step of the research, two strategies were explored for the removal of Stenotrophomonas maltophilia biofilms. Firstly, the importance of incorporating a cleaning step for the removal of biofilms was further characterized by microscopical analysis and (partial) EPS characterization. This analysis revealed that incorporating a cleaning step induced a higher EPS removal and inhibition of the α-polysaccharides production during regrowth. Based on these results, optimization of C&D was attempted, revealing that optimal S. maltophilia biofilm removal and inhibition of the regrowth of the residual biofilm was not achieved by increasing the concentration of the cleaning agent but by increasing the disinfectant concentration after a cleaning step with the lowest concentration. Secondly, we demonstrated the potential of different modified surfaces to reduce the biofilm formation and/or facilitating the biofilm removal of Stenotrophomonas maltophilia.status: publishe

Identification and Spoilage Potential of the Remaining Dominant Microbiota on Food Contact Surfaces after Cleaning and Disinfection in Different Food Industries

After cleaning and disinfection (C&D), surface contamination can still be present in the production environment of food companies. Microbiological contamination on cleaned surfaces can be transferred to the manufactured food and consequently lead to foodborne illness and early food spoilage. However, knowledge about the microbiological composition of residual contamination after C&D and the effect of this contamination on food spoilage is lacking in various food sectors. In this study, we identified the remaining dominant microbiota on food contact surfaces after C&D in seven food companies and assessed the spoilage potential of the microbiota under laboratory conditions. The dominant microbiota on surfaces contaminated at ≥102 CFU/100 cm2 after C&D was identified based on 16S rRNA sequences. The ability of these microorganisms to hydrolyze proteins, lipids, and phospholipids, ferment glucose and lactose, produce hydrogen sulfide, and degrade starch and gelatin also was evaluated. Genera that were most abundant among the dominant microbiota on food contact surfaces after C&D were Pseudomonas, Microbacterium, Stenotrophomonas, Staphylococcus, and Streptococcus. Pseudomonas spp. were identified in five of the participating food companies, and 86.8% of the isolates evaluated had spoilage potential in the laboratory tests. Microbacterium and Stenotrophomonas spp. were identified in five and six of the food companies, respectively, and all tested isolates had spoilage potential. This information will be useful for food companies in their quest to characterize surface contamination after C&D, to identify causes of microbiological food contamination and spoilage, and to determine the need for more thorough C&D.status: publishe

Identification and spoilage potential of the remaining dominant microbiota on food contact surfaces after cleaning and disinfection in different food industries

After cleaning and disinfection (C&D), surface contamination can still be present in the production environment of food companies. Microbiological contamination on cleaned surfaces can be transferred to the manufactured food and consequently lead to foodborne illness and early food spoilage. However, knowledge about the microbiological composition of residual contamination after C&D and the effect of this contamination on food spoilage is lacking in various food sectors. In this study, we identified the remaining dominant microbiota on food contact surfaces after C&D in seven food companies and assessed the spoilage potential of the microbiota under laboratory conditions. The dominant microbiota on surfaces contaminated at >= 10(2) CFU/100 cm(2) after C&D was identified based on 16S rRNA sequences. The ability of these microorganisms to hydrolyze proteins, lipids, and phospholipids, ferment glucose and lactose, produce hydrogen sulfide, and degrade starch and gelatin also was evaluated. Genera that were most abundant among the dominant microbiota on food contact surfaces after C&D were Pseudomonas, Microbacterium, Stenotrophomonas, Staphylococcus, and Streptococcus. Pseudomonas spp. were identified in five of the participating food companies, and 86.8% of the isolates evaluated had spoilage potential in the laboratory tests. Microbacterium and Stenotrophomonas spp. were identified in five and six of the food companies, respectively, and all tested isolates had spoilage potential. This information will be useful for food companies in their quest to characterize surface contamination after C&D, to identify causes of microbiological food contamination and spoilage, and to determine the need for more thorough C&D

Evaluation of two surface sampling methods for microbiological and chemical analyses to assess the presence of biofilms in food companies

Biofilms are an important source of contamination in food companies, yet the composition of biofilms in practice is still mostly unknown. The chemical and microbiological characterization of surface samples taken after cleaning and disinfection is very important to distinguish free-living bacteria from the attached bacteria in biofilms. In this study, sampling methods that are potentially useful for both chemical and microbiological analyses of surface samples were evaluated. In the manufacturing facilities of eight Belgian food companies, surfaces were sampled after cleaning and disinfection using two sampling methods: the scraper–flocked swab method and the sponge stick method. Microbiological and chemical analyses were performed on these samples to evaluate the suitability of the sampling methods for the quantification of extracellular polymeric substance components and microorganisms originating from biofilms in these facilities. The scraper–flocked swab method was most suitable for chemical analyses of the samples because the material in these swabs did not interfere with determination of the chemical components. For microbiological enumerations, the sponge stick method was slightly but not significantly more effective than the scraper–flocked swab method. In all but one of the facilities, at least 20% of the sampled surfaces had more than 102 CFU/100 cm2. Proteins were found in 20% of the chemically analyzed surface samples, and carbohydrates and uronic acids were found in 15 and 8% of the samples, respectively. When chemical and microbiological results were combined, 17% of the sampled surfaces were contaminated with both microorganisms and at least one of the analyzed chemical components; thus, these surfaces were characterized as carrying biofilm. Overall, microbiological contamination in the food industry is highly variable by food sector and even within a facility at various sampling points and sampling times

Functional probiotic products based on fermented wort with Lactobacillus rhamnosus GG and Lactobacillus casei

status: accepte

Biofilms of Stenotrophomonas maltophilia protect Lactobacillus brevis food spoilage bacteria against classical cleaning and disinfection protocols

edition: Biofilms8status: publishe

Development of a Simultaneous Saccharificationand Fermentation process using alkaline pretreated wheat straw

status: accepte