Occurrence and characterisation of residual contamination and biofilms in food processing environments and poultry drinking water systems

Despite a regular cleaning and disinfection (C&D), residual bacterial contamination and biofilms still occur in food companies and primary animal production environments. In case pathogenic and spoilage organisms remain, these could lead to early food spoilage, foodborne illness and pose a threat for animal and human health. Further knowledge concerning the occurrence, composition and characteristics is needed to have a better estimate on the impact of this residual contamination. Therefore, this research aimed at gaining insights in the presence and characteristics of residual bacterial contamination and biofilms in different food sectors and in primary animal production, more specifically the drinking water system (DWS) in broiler houses.

In the first part of the research (chapters 3 and 4), biofilms and residual bacterial contamination in eight food companies of different sectors were mapped and investigated. To do so, there was a need for a suitable sampling method that allowed detection and/or quantification of microorganisms and biofilm matrix components. Two surface sampling methods were tested, the sponge stick method and the scraper-flocked swab method, whereby the latter method was evaluated as the most suitable. Sampling with this method provided results of the bacterial load and chemical composition of surface contamination after C&D. Bacterial enumerations were on average 3.62 ± 1.20 log CFU/100 cm² but reached up to 7.23 log CFU/100 cm². Respectively 20%, 15% and 8% of the surfaces investigated for biofilm matrix components were contaminated with low quantities of proteins, carbohydrates and uronic acids. On 17% of the investigated surfaces, both microorganisms and at least one of the chemical compounds were found, which is an indication for the presence of biofilm. The presence and the degree of residual contamination is highly variable by food sector, food company, sampling point and even sampling time. Genera that were most abundant in the residual bacterial contamination on food contact surfaces after C&D in the different food companies were Pseudomonas, Microbacterium and Stenotrophomonas, however 60% of the identified genera were company-specific. Of all the evaluated isolates, 88% had some kind of spoilage potential, with the ability to break down lipids as the most prevalent property. However it remains difficult to estimate the possible impact of these microorganisms on food safety and spoilage since it is not known whether they will: be transferred from the surface to the food products, survive and grow in the food products and consequently cause spoilage.

In the second part of the research (chapters 5 and 6), the focus was on residual contamination and biofilm formation on the inside of the DWS in broiler houses after disinfection. Water quality in the DWS plays an important role in the general health and performance of broiler chickens since pathogens might be present. Conditions in the DWS of broilers are ideal for microbial biofilm formation. The presence of this contamination on the inside of the DWS was assessed in terms of bacterial load and chemical composition. Average bacterial counts of 6.03 ± 1.53 log CFU/20cm² were observed, ranging up to 9.00 log CFU/20cm² at some points. Proteins, carbohydrates and uronic acids were again found in low quantities in 58%, 14% and 5% of the samples, respectively. On 63% of the investigated surfaces, the presence of biofilm was suspected since microorganisms were detected in combination with at least one of the analysed chemical components. The most identified dominant species in the DWS were Stenotrophomonas maltophilia, Pseudomonas geniculata and Pseudomonas aeruginosa, which are opportunistic human pathogens. 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 in 96-well microtiter plates. Finally, the hypothesis that commensal bacteria in biofilms on surfaces could prevent attachment of pathogens such as Salmonella spp. was investigated. Since there is an increasing problem with Salmonella Java contamination on broiler chickens in Belgium, this pathogen was used to investigate its interaction with the commensal Pseudomonas putida, which is part of the natural microbiota in the DWS. Therefore, a new model that simulates biofilm formation on the inside of the DWS was developed and validated. In this model, Salmonella Java was evaluated as a strong biofilm former. However, when applied in the presence of Pseudomonas putida, biofilm formation by Salmonella Java was reduced due to competitive interactions indicating the potential of Pseudomonas putida as a biocontrol agent.

In conclusion, this research provided interesting new information for food companies to be used in their fight against unwanted contamination and for the development of more efficient C&D procedures. Also in primary animal production, new insights concerning the presence and composition of DWS contamination and its role in the prevention of pathogens were obtained.