Occurrence and characterization of biofilms in drinking water systems of broiler houses

Introduction
A lot of bacteria are able to attach and grow on almost every surface forming complex microbial communities termed biofilms. Since they are highly resistant to multiple stress conditions and disinfectants, biofilms have become a concerned source of contamination in the agro-food chain. The importance and role of biofilms in broiler houses and more specifically in drinking water systems is however insufficiently known. Detection and characterization of biofilms in the drinking water system of broiler houses can help to provide new insights in this issue. The aim of this study was to evaluate the presence of biofilms in the drinking water system of different broiler houses and to characterize the microbial population of these presumptive biofilms.
Material and methods
In 5 different broiler farms, surfaces on the inside of the drinking water system were sampled after disinfection using a flocked swab(Copan). Sampling points mostly considered the end of the pipes, openings at the level of drinking cups and the inside of pressure regulators. In total, 85 surface samples were taken from surface areas of 20cm². Beside total aerobic count and counts for Pseudomonas spp. also chemical analyses (proteins, sugars and uronic acids ) were performed. After microbiological enumerations, morphologically different colonies were isolated. The isolated dominant bacteria were identified using (GTG)5 clustering followed by 16S rRNA gene sequencing. The possibility of the collected dominant bacteria to form biofilm under lab conditions was evaluated in 96-well microtiter plates.
Results and discussion
All of the 85 sampled surfaces in the drinking water system were contaminated with 1.9 to 9.0 log CFU/20cm² for total aerobic counts. Counts for Pseudomonas spp. were on average 0.9 log CFU/20cm² lower, indicating the important presence of this genus. When chemical and microbiological results were combined, 63% of the sampled surfaces appeared to be contaminated with both micro-organisms and at least one of the with biofilm associated chemical components and consequently identified as carrying biofilm. Micro-organisms were mostly found in combination with proteins (58% of the surfaces). A wide diversity of predominantly Gram negative micro-organisms were identified, most of them were farm-dependent. Only Stenotrophomonas maltophilia was found in all 5 broiler farms. Moreover this species was present in high numbers (≥ 4.0 log CFU/20cm²). Pseudomonas aeruginosa and Pseudomonas hibiscicola were found in all but one of the sampled farms. Isolates with the strongest biofilm forming capacities belonged to the genus Pseudomonas and Stenotrophomonas.
Conclusion
On all the sampled surfaces in the drinking water systems, high numbers of micro-organisms were found even after disinfection. The most identified species were Stenotrophomonas maltophilia, Pseudomonas geniculata and Pseudomonas aeruginosa. Moreover, most of the isolates identified as these species were classified as strong biofilm formers. Though, differences in biofilm forming capacities on species and strain level were observed.