Potential of Flow Cytometric Approaches for Rapid Microbial Detection and Characterization in the Food Industry-A Review.

As microbial contamination is persistent within the food and bioindustries and foodborne infections are still a significant cause of death, the detection, monitoring, and characterization of pathogens and spoilage microorganisms are of great importance. However, the current methods do not meet all relevant criteria. They either show (i) inadequate sensitivity, rapidity, and effectiveness; (ii) a high workload and time requirement; or (iii) difficulties in differentiating between viable and non-viable cells. Flow cytometry (FCM) represents an approach to overcome such limitations. Thus, this comprehensive literature review focuses on the potential of FCM and fluorescence in situ hybridization (FISH) for food and bioindustry applications. First, the principles of FCM and FISH and basic staining methods are discussed, and critical areas for microbial contamination, including abiotic and biotic surfaces, water, and air, are characterized. State-of-the-art non-specific FCM and specific FISH approaches are described, and their limitations are highlighted. One such limitation is the use of toxic and mutagenic fluorochromes and probes. Alternative staining and hybridization approaches are presented, along with other strategies to overcome the current challenges. Further research needs are outlined in order to make FCM and FISH even more suitable monitoring and detection tools for food quality and safety and environmental and clinical approaches.

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

Biofilm characteristics of Microbacterium lacticum D84 (M. lacticum) and Staphylococcus capitis subsp. capitis (S. capitis) on polytetrafluoroethylene and AISI-304 stainless steel at early- (24, 48 h) and late-stage (144, 192 h) biofilm formation were investigated. M. lacticum biofilm structure was more developed compared to S. capitis, representing vastly mature biofilms with a strongly developed amorphous matrix, possibly extracellular polymeric substances (EPSs), at late-stage biofilm formation. S. capitis showed faster growth behavior but still resulted in a relatively flat biofilm structure. Strong correlations were found between several roughness parameters and S. capitis surface coverage (r ≥ 0.98), and between total surface free energy (γs) and S. capitis surface coverage (r = 0.89), while M. lacticum remained mostly unaffected. The pronounced ubiquitous biofilm characteristics make M. lacticum D84 a suitable model for biofilm research. Studying biofilm formation of these bacteria may help one understand bacterial adhesion on interfaces and hence reduce biofilm formation in the food industry.

The Influence of Meat Batter Composition and SausageDiameter on Microbiota and Sensory Traits of ArtisanalWild Boar Meat Sausages.

In this study, the influence of meat batter composition and sausage diameter on the development of microbiota and sensory traits of traditional, spontaneously fermented wild boar meat sausages are evaluated. This research also demonstrates how principal component analysis (PCA) can be used to relate product sensory properties to particular microbial genotype and to select potential starter or adjunct culture. Generally, similar microbiological results were obtained in all types of products. The undesirable microbiota was either not detected at any sausage production stage or its number decreased below the detection limit in ripened sausages. The low growth rate of lactic acid bacteria (LAB) was consistent with the obtained pH and slow acidification rate. Although no differences in the composition of LAB species were noticed between sausage types (50S=50% wild boar meat in small casing, 50L=50% wild boar meat in large casing, 100S=100% wild boar meat in small casing), a clear separation based on LAB genotypes could be observed. Upon quantitative descriptive analysis, significant differences in sensory attributes between sausage types were established. According to the PCA, the overall acceptability traits of sausages are closely linked to one Leuconostoc mesenteroides genotype (LM_4). Of all tested technological properties, LM_4 strains showed remarkable acidification ability, lowering the pH from pH=5.41 to 3.74, and pronounced proteolytic activity on skimmed milk as well as antagonistic activity against Staphylococcus aureus (DSM 20231) and Brochothrix thermosphacta (LMG 17208). Lipolytic and haemolytic activities were not detected, and all analyzed strains were susceptible to tested antibiotics and possessed no biogenic amine genes.

Assessment of Microbial Community Dynamics in River Bank Filtrate Using High-Throughput Sequencing and Flow Cytometry.

Surface-groundwater interactions play an important role in microbial community compositions of river bank filtrates. Surface water contaminations deriving from environmental influences are attenuated by biogeochemical processes in the hyporheic zone, which are essential for providing clean and high-quality drinking water in abstraction wells. Characterizing the flow regime of surface water into the groundwater body can provide substantial information on water quality, but complex hydraulic dynamics make predictions difficult. Thus, a bottom up approach using microbial community shifting patterns as an overall outcome of dynamic water characteristics could provide more detailed information on the influences that affect groundwater quality. The combination of high-throughput sequencing data together with flow cytometric measurements of total cell counts reveals absolute abundances among taxa, thus enhancing interpretation of bacterial dynamics. 16S rRNA high-throughput sequencing of 55 samples among six wells in a well field in Austria that is influenced by river bank filtrate within a time period of 3 months has revealed both, clear differences as well as strong similarity in microbiome compositions between wells and dates. A significant community shift from April to May occurred in four of six wells, suggesting that surface water flow regimes do affect these wells stronger than others. Triplicate sampling and subsequent sequencing of wells at different dates proved the method to be reproducible. Flow cytometric measurements of total cells indicate microbial shifts due to increased cell counts and emphasize the rise of allochthonous microorganisms. Typical freshwater bacterial lineages (Verrucomicrobia, Bacteroidetes, Actinobacteria, Cyanobacteria, Armatimonadetes) were identified as most increasing phyla during community shifts. The changes are most likely a result of increased water abstraction in the wells together with constant river water levels rather than rain events. The results provide important knowledge for future implementations of well utilization in dependency of the nearby Danube River water levels and can help drawing conclusions about the influence of surface water in the groundwater such that hygienically save and clean drinking water with a stable microbial community can be provided.

Sublethal Injury and Viable but Non-culturable (VBNC) State in Microorganisms During Preservation of Food and Biological Materials by Non-thermal Processes.

The viable but non-culturable (VBNC) state, as well as sublethal injury of microorganisms pose a distinct threat to food safety, as the use of traditional, culture-based microbiological analyses might lead to an underestimation or a misinterpretation of the product's microbial status and recovery phenomena of microorganisms may occur. For thermal treatments, a large amount of data and experience is available and processes are designed accordingly. In case of innovative inactivation treatments, however, there are still several open points with relevance for the investigation of inactivation mechanisms as well as for the application and validation of the preservation processes. Thus, this paper presents a comprehensive compilation of non-thermal preservation technologies, i.e., high hydrostatic pressure (HHP), pulsed electric fields (PEFs), pulsed light (PL), and ultraviolet (UV) radiation, as well as cold plasma (CP) treatments. The basic technological principles and the cellular and molecular mechanisms of action are described. Based on this, appropriate analytical methods are outlined, i.e., direct viable count, staining, and molecular biological methods, in order to enable the differentiation between viable and dead cells, as well as the possible occurrence of an intermediate state. Finally, further research needs are outlined.

Increased Isoprenoid Quinone Concentration Modulates Membrane Fluidity in Listeria monocytogenes at Low Growth Temperatures.

Listeria monocytogenes is a food pathogen capable of growing at a broad temperature range from 50°C to refrigerator temperatures. A key requirement for bacterial activity and growth at low temperatures is the ability to adjust the membrane lipid composition to maintain cytoplasmic membrane fluidity. In this study, we confirmed earlier findings that the extents of fatty acid profile adaptation differed between L. monocytogenes strains. We were able to demonstrate for isolates from food that growth rates at low temperatures and resistance to freeze-thaw stress were not impaired by a lower adaptive response of the fatty acid composition. This indicated the presence of a second adaptation mechanism besides temperature-regulated fatty acid synthesis. For strains that showed weaker adaptive responses in their fatty acid profiles to low growth temperature, we could demonstrate a significantly higher concentration of isoprenoid quinones. Three strains even showed a higher quinone concentration after growth at 6°C than at 37°C, which is contradictory to the reduced respiratory activity at lower growth temperatures. Analyses of the membrane fluidity in vivo by measuring generalized polarization and anisotropy revealed modulation of the transition phase. Strains with increased quinone concentrations showed an expanded membrane transition phase in contrast to strains with pronounced adaptations of fatty acid profiles. The correlation between quinone concentration and membrane transition phase expansion was confirmed by suppression of quinone synthesis. A reduced quinone concentration resulted in a narrower transition phase. Expansion of the phase transition zone by increasing the concentration of non-fatty acid membrane lipids is discussed as an additional mechanism improving adaptation to temperature shifts for L. monocytogenes strains.IMPORTANCEListeria monocytogenes is a foodborne pathogen with an outstanding temperature range for growth. The ability for growth at temperatures close to the freezing point constitutes a serious contamination potential for cold stored food. The only known mechanism of the species for adaptation of membrane fluidity is modification of the membrane fatty acid composition. We were able to demonstrate that, at least for some strains, this adaptation mechanism is supported by regulation of the menaquinone concentration. The increase of this neutral membrane lipid is correlated with fluidization of the membrane under low-temperature conditions and therefore represents a fatty acid-independent mechanism for adaptation to low temperatures.