7th joint conference "Joint Microbiology & Infection Conference 2024" of the DGHM and VAAM

Prof. Dr.

Chair

Biocontrol of pseudomonas biofilms in dairy farming environments using bacteriophages

Food microbiology and -hygiene

IntroductionPseudomonads are a genus of ubiquitous Gram-negative bacteria that encompasses species with major impact on food spoilage, especially in the dairy industry. Several physiological properties enable them to persist in production plants and increase the risk of food spoilage: Pseudomonas bacteria are psychrophile and outgrow other bacteria in cool conditions that are often present during storage of fresh produce. Moreover, they form biofilms, allowing them to persist in food plants and efficiently resist sanitation measures. Lastly, a major problem especially in the dairy industry is the ability of some Pseudomonas species to produce heat-stable proteases, which can withstand procedures like ultra-high-temperature treatment of raw milk, causing the decay of milk proteins even after sterilization, and thus leading to premature spoilage of the milk. In order to eliminate Pseudomonas species relevant for milk spoilage, we explore the application of bacteriophages (viruses that infect bacteria) to eradicate persistent biofilms and limit the contamination of milk with heat-stable proteases.GoalsThe goal of this study is to identify potent phages that are active against a broad spectrum of relevant spoilage species like P. proteolytica, P. lactis and P. protegens. The combined use of those phages in a cocktail will be utilized to eradicate Pseudomonas biofilms, which are grown under practically relevant conditions.ResultsA total of 29 phages were isolated. The majority (25) belonged to the Caudoviricetes class, which was expected as it is most prevalent in nature. Within that class, the majority of the isolated phages has not been described in literature. The host range varied significantly and some phages infected as much as 19 host strains, making them interesting candidates for biofilm inhibition. Their efficiency in the eradication of biofilms will be evaluated in coming experiments.SummaryThe use of phages as biocontrol agents against Pseudomonas biofilms is explored in this study and includes the identification of phages infecting relevant bacterial strains and the evaluation of their efficiency to degrade their host&quot;s biofilms.

Oral Presentation

Attachment of Listeria monocytogenes to corn salad.

Question L. monocytogenes is one of the most important foodborne pathogens because of the high hospitalization and mortality rate. Little is known about how exactly L. monocytogenes attaches to corn salad, one of the most prevalent fresh produce. Pathogenicity is highly serovar dependent giving rise to the question if there is a link between colonization and virulence. After contamination, attachment and biofilm formation poses a serious risk because it allows the bacteria to persist over long time periods. Therefore, the aim of this study is to investigate the attachment and colonization of L. monocytogenes on corn salad as well as its ability to form biofilm and to answer the question which genes are involved in these processes.Methods Three strains of L. monocytogenes strains isolated from plant foods as well as from clinical samples were first characterized genotypically using whole genome sequencing (WGS). Strain NCTC 10587 was fluorescently labelled using the chromosomally integrating plasmid vector pAD1. Confocal laser scanning microscopy of the fluorescently labelled strain was used to confirm attachment to leaf surfaces, scanning electron microscopy (SEM) was used to track attachment at the single cell level. To quantify the biofilm formation, in vitro crystal violet assays were performed in triplicate in three different media at two temperatures for 72 h.Results WGS of the three L. monocytogenes strains confirmed the presence of major virulence factors. Fluorescence microscopy was performed with infected corn salad leaves 48 h post infection. SEM imaging showed L. monocytogenes mainly colonizing the stomata of corn salad and no biofilm formation was confirmed on the surrounding areas. Contrary, crystal violet assays showed that biofilm formation was highest in Luria Bertani medium at 22°C after 12 to 14 h.Conclusion The results of this study suggest a colonization of the corn salad stomata without biofilm formation on the leaf surfaces, even though the strains are capable of biofilm formation in vitro. Further studies need to elucidate the relevance of these findings for consumer safety.

Parameters influencing water kefir grain growth dynamics by image analysis

Water kefir is a fermented beverage that is produced by adding water kefir grains to a sucrose solution with dried fruits. The approximately 1-10 mm large grains consist of a bacterial exopolysaccharide matrix and are mainly colonised by lactic acid bacteria, acetic acid bacteria and yeasts. The production depends on the successful cultivation and propagation of the existing grains: During fermentation cycles, the mass of the grains increases, but growth dynamics between the start and end points of fermentation and the parameters that can influence this process have not yet been analysed. Monitoring growth is potentially relevant for the industrial production of water kefir, as no defined starter cultures are available. Our aim was therefore to establish a method for the continuous documentation of grain growth and thus enable a kinetic description of the involved processes. We developed a documentation system that takes high quality pictures, which are then further processed with Fiji image-analysis software. Kefir grains were selected by size and incubated in standardized water kefir medium at room temperature for 72 h. The number of and increase in white pixels in the pictures against a black background represent a measure of the two-dimensional grain size and growth, respectively. The developed method enables the visualization of grain growth curves as a function of white pixels over fermentation time. These are comparable to classic bacterial growth curves, as they exhibit lag, exponential and stationary phases. The influence of the number of starter grains and their size could be evaluated based on curve progression and total growth. It was found that an increasing number of grains and larger grain sizes have a negative effect on the overall growth rate. However, the exponential and stationary growth phases tend to be reached faster as the number of grains increases. In ongoing experiments, the influence of the sugar content and the type of water in the medium is being investigated.

Oral Session
Food Microbiology and -Hygiene

Appointment

Chair

Topic

Programme

Oral SessionFood Microbiology and -Hygiene

Appointment

Chair

Topic

Programme

Oral Session
Food Microbiology and -Hygiene