Bacterial community composition of biofilms in milking machines of two dairy farms assessed by a combination of culture-dependent and –independent methods

Abstract

Dairy biofilms as a source of contamination of milk and its products are of great concern in the dairy industry. For a reliable risk assessment, knowledge about the microbial community composition of biofilms in the milking systems of dairy farms must be improved. In this work, swab samples of milking machine biofilms of two dairy farms were investigated by a combination of culture-dependent and -independent methods. Spots in the milking system with enhanced microbial colonization were identified by quantification on selective and non-selective media. In addition, stainless steel coupons were placed into the piping system of a milking machine, removed after several milking intervals, and investigated for colonization by cultivation and culture-independently. Isolates were differentiated and identified by a combination of chemotaxonomical methods and 16S rRNA sequencing. The culture-independent approach involved treatment of the samples with the viability dye propidium monoazide prior to direct DNA-extraction by enzymatic cell lysis and cloning to exclude bias from dead biomass. The milking equipment retainers and the outlet of the milk bulk tank were identified as highly colonized spots on both farms. A high bacterial diversity was detected covering the phyla Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria. Presence of biofilms was demonstrated on several materials including stainless steel and plastic, which are frequently used in milking machines, but also in dairy processing plants. Growth of mainly Gram-positive bacteria with high percentages of the phylum Actinobacteria was detected on the stainless steel coupons after exposition in the milking system for two to three days. Knowledge about the heterogenic microbial load on different parts of the milking machines and the stainless steel coupons will help to identify primary colonizers of the milking system, to assess the risk potential of biofilms for raw milk, to improve sanitation processes and to identify parts of the milking machine, which should be improved by hygienic design.

Biofilm-Forming Ability of Microbacterium lacticum and Staphylococcus capitis Considering Physicochemical and Topographical Surface Properties

“…As biofilms show increased resistance to mechanical, physsical, and chemical treatments, in comparison to their planktonic state [ 7 ], it is crucial to select suitable sanitation methods [ 8 ]. It is vital to understand that the embedded extracellular matrix of persistent biofilms can protect present potential pathogens and spoilage microorganisms from disinfection treatments [ 9 ]. Any persistent microorganism with the ability to form biofilms can lead to human diseases or alteration in sensory characteristics [ 4 ].…”

“…Besides staphylococci, Microbacterium spp. were previously isolated from dairy, egg, ready-to-eat, and meat-producing industries [ 9 , 19 , 20 ], among which M. lacticum is abundant on food contact surfaces in dairy-related facilities [ 9 , 19 ]. Microbacterium spp.…”

“…M. lacticum adherent to abiotic surfaces is not only found to be persistent against cleaning and disinfection [ 9 ] but also against pasteurization [ 20 ]. The study by Weber, Liedtke, Plattes and Lipski [ 9 ] also reported that isolated M. lacticum can lead to contamination and reduced shelf-life of ultra-high-temperature (UHT) processed milk, due to its spoilage- and biofilm-forming potential. Both S. capitis and M. lacticum were previously studied in their planktonic form, but there was no detailed study on their biofilm-related characteristics.…”

Insights into the Microbiological Safety of Wooden Cutting Boards Used for Meat Processing in Hong Kong’s Wet Markets: A Focus on Food-Contact Surfaces, Cross-Contamination and the Efficacy of Traditional Hygiene Practices

“…These authors also observed that some of these bacterial species were resistant to certain types of disinfectants [55], which implies that robust, well-validated hygiene regimes should be used for cleaning food processing environments. Weber et al [56] studied the bacterial composition of biofilms in bovine milk and found that Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes were the dominant phyla. In the phylum Actinobacteria, Rhodococcus, Dermacoccus, Gordonia, Kocuria and Microbacterium were the main genera.…”

Phenotypic and Genotypic Investigation of Two Representative Strains of Microbacterium Species Isolated From Micro-Filtered Milk: Growth Capacity and Spoilage-Potential Assessment

“…However, it has been shown that from the time of contamination until the end of the shelf life of microfiltered milk, Microbacterium's proteolytic and lipolytic activities are below the threshold of detection (Schmidt et al, 2012). Regarding dairy products, it is interesting to note that strains of the genus Microbacterium are often found on the rind of smear-ripened cheeses (Mounier et al, 2005(Mounier et al, , 2007 and that the community of psychrophilic bacteria in raw milk, including Microbacterium, has the ability to form biofilms along the whole milk chain (e.g., on the collection and storage equipment), which poses a risk to the stability of the milk (Weber et al, 2019). However, no data are available on the genomic structures underlying the phenotypes of the genus Microbacterium that are involved in the preservation of extended shelf-life (ESL) milk.…”