Gaps in the assortment of rapid assays for microorganisms of interest to the dairy industry.

This review presents the results of a study into the offering of rapid microbial detection assays to the Irish dairy industry. At the outset, a consultation process was undertaken whereby key stakeholders were asked to compile a list of the key microorganisms of interest to the sector. The resultant list comprises 19 organisms/groups of organisms divided into five categories: single pathogenic species (Cronobacter sakazakii, Escherichia coli and Listeria monocytogenes); genera containing pathogenic species (Bacillus, Clostridium, Listeria, Salmonella; Staphylococcus); broad taxonomic groupings (Coliforms, Enterobacteriaceae, fecal Streptococci, sulfite reducing bacteria/sulfite reducing Clostridia [SRBs/SRCs], yeasts and molds); organisms displaying certain growth preferences or resistance as regards temperature (endospores, psychrotrophs, thermodurics, thermophiles); indicators of quality (total plate count, Pseudomonas spp.). A survey of the rapid assays commercially available for the 19 organisms/groups of organisms was conducted. A wide disparity between the number of rapid tests available was found. Four categories were used to summarize the availability of rapid assays per organism/group of organisms: high coverage (>15 assays available); medium coverage (5-15 assays available); low coverage (<5 assays available); no coverage (0 assays available). Generally, species or genera containing pathogens, whose presence is regulated-for, tend to have a good selection of commercially available rapid assays for their detection, whereas groups composed of heterogenous or even undefined genera of mainly spoilage organisms tend to be "low coverage" or "no coverage." Organisms/groups of organisms with "low coverage" by rapid assays include: Clostridium spp.; fecal Streptococci; and Pseudomonas spp. Those with "no coverage" by rapid assays include: endospores; psychrotrophs; SRB/SRCs; thermodurics; and thermophiles. An important question is: why have manufacturers of rapid microbiological assays failed to respond to the necessity for rapid methods for these organisms/groups of organisms? The review offers explanations, ranging from the technical difficulty involved in detecting as broad a group as the thermodurics, which covers the spores of multiple sporeforming genera as well at least six genera of mesophilic nonsporeformers, to the taxonomically controversial issue as to what constitutes a fecal Streptococcus or SRBs/SRCs. We review two problematic areas for assay developers: validation/certification and the nature of dairy food matrices. Development and implementation of rapid alternative test methods for the dairy industry is influenced by regulations relating to both the microbiological quality standards and the criteria alternative methods must meet to qualify as acceptable test methods. However, the gap between the certification of developer's test systems as valid alternative methods in only a handful of representative matrices, and the requirement of dairy industries to verify the performance of alternative test systems in an extensive and diverse range of dairy matrices needs to be bridged before alternative methods can be widely accepted and adopted in the dairy industry. This study concludes that many important dairy matrices have effectively been ignored by assay developers.

Protein A-Mediated Binding of Staphylococcus spp. to Antibodies in Flow Cytometric Assays and Reduction of This Binding by Using Fc Receptor Blocking Reagent.

Staphylococcus aureus and other coagulase-positive Staphylococcus spp. bind the Fc region of IgG antibodies through expression of protein A (SpA). These species have consequently been a source of false-positive signals in antibody-based assays designed to detect other target bacteria. Here, flow cytometry was used to study the influence of a number of factors on the SpA-mediated binding of single cells to an anti-human IgG antibody, including strain, heat killing, overnight storage, growth phase, cell physiology, surface adhesion, and growth in model food systems. Through the costaining of antibody-stained cells with the permeability dye propidium iodide and calcein violet AM, the cell physiological status was related to SpA-mediated antibody binding. Generally, permeabilized cells lacking esterase activity did not strongly bind antibody. The binding of a number of commercially available polyclonal IgG antibodies to non-Staphylococcus spp. was also characterized. Not all SpA-expressing species showed strong binding of mouse IgG, and one species not known to express SpA showed strong binding. Most SpA-expressing strains bound rabbit IgG antibodies to some extent, whereas only one strain bound goat IgG. To reduce or eliminate SpA-mediated IgG binding, the following products were evaluated as blocking reagents and applied prior to staining with primary or secondary antibody: normal rabbit serum, mouse IgG isotype control, goat IgG, and a commercial FcR blocking reagent. Only the FcR blocking reagent consistently reduced SpA-mediated binding of Staphylococcus spp. to antibodies against other species and could be recommended as a blocking reagent in immunoassays designed to detect non-Staphylococcus species.IMPORTANCE This study characterizes a widespread but little-studied problem associated with the antibody-based detection of microbes-the Staphylococcus protein A (SpA)-mediated binding of IgG antibodies-and offers a solution: the use of commercial FcR blocking reagent. A common source of false-positive signals in the detection of microbes in clinical, food, or environmental samples can be eliminated by applying this study's findings. Using flow cytometry, the authors demonstrate the extent of heterogeneity in a culture's SpA-mediated binding of antibodies and that the degree of SpA-mediated antibody binding is strain, growth phase, and food matrix dependent and influenced by simulated food processing treatments and cell adherence. In addition, our studies of SpA-mediated binding of Staphylococcus spp. to antibodies against other bacterial species produced a very nuanced picture, leading us to recommend testing against multiple strains of S. aureus and S. hyicus of all antibodies to be incorporated into any immunoassay designed to detect a non-Staphylococcus spp.

Heat and Chemical Treatments Affect the Viability, Morphology, and Physiology of Staphylococcus aureus and Its Subsequent Antibody Labeling for Flow Cytometric Analysis.

The effects of heat and chemical treatments on Staphylococcus aureus viability and physiology and their subsequent effects on antibody binding ability and cell morphology were measured. Treatments included lethal and sublethal heat; exposure to organic acids, salt, and sodium hydroxide; and freeze-thawing. Strain-related differences in viability were noted depending on treatment and were reflected in changes in physiology as monitored by flow cytometry (FCM) using three different staining protocols: SYTO 9/propidium iodide (PI), DiOC2(3), or calcein acetoxymethyl ester (calcein-AM)/PI. Treatments that resulted in significant losses in viability as measured by plate counting were reflected better by the first two staining combinations, as intracellular calcein-AM uptake may have been impaired by certain treatments. FCM analysis using labeling by commercial anti-S. aureus antibodies indicated that differences in cell physiology as a result of treatments influenced immunofluorescence detection. The ratio of the mean fluorescence intensities of stained cells to those of unstained cells [MFI/MFI(us)] varied with treatment, five of these treatments, including freeze-thaw, citric acid, oxalic acid, NaCl, and NaOH treatments, resulted in significantly lower fluorescence values compared to controls.IMPORTANCE FCM data indicated that cells conventionally considered to be dead and which would not give rise to CFU in a plate count assay, e.g., cells heated to 80°C, were labeled by antibody staining. This finding suggests that without the inclusion of a live/dead discriminating dye, these cells would be erroneously detected as viable within an FCM assay. Reductions in antibody staining due to physicochemical treatment were strain related, reflecting the complexity of the phenomenon under study and illustrating that substantial validation of any new antibody detection-based method, including physiological staining and cell sorting, should be undertaken. Researchers should be aware of physicochemical treatments causing false-negative results: in this study, freeze-thawing severely reduced antibody binding without affecting the viability of a substantial percentage of cells. Scanning electron microscopy carried out on treated cells revealed a range of morphological changes resulting from physicochemical treatments which may have hindered antibody binding.

MiR-93 Controls Adiposity via Inhibition of Sirt7 and Tbx3.

Conquering obesity has become a major socioeconomic challenge. Here, we show that reduced expression of the miR-25-93-106b cluster, or miR-93 alone, increases fat mass and, subsequently, insulin resistance. Mechanistically, we discovered an intricate interplay between enhanced adipocyte precursor turnover and increased adipogenesis. First, miR-93 controls Tbx3, thereby limiting self-renewal in early adipocyte precursors. Second, miR-93 inhibits the metabolic target Sirt7, which we identified as a major driver of in vivo adipogenesis via induction of differentiation and maturation of early adipocyte precursors. Using mouse parabiosis, obesity in mir-25-93-106b(-/-) mice could be rescued by restoring levels of circulating miRNA and subsequent inhibition of Tbx3 and Sirt7. Downregulation of miR-93 also occurred in obese ob/ob mice, and this phenocopy of mir-25-93-106b(-/-) was partially reversible with injection of miR-93 mimics. Our data establish miR-93 as a negative regulator of adipogenesis and a potential therapeutic option for obesity and the metabolic syndrome.

Development of defined microbial population standards using fluorescence activated cell sorting for the absolute quantification of S. aureus using real-time PCR.

In this article, four types of standards were assessed in a SYBR Green-based real-time PCR procedure for the quantification of Staphylococcus aureus (S. aureus) in DNA samples. The standards were purified S. aureus genomic DNA (type A), circular plasmid DNA containing a thermonuclease (nuc) gene fragment (type B), DNA extracted from defined populations of S. aureus cells generated by Fluorescence Activated Cell Sorting (FACS) technology with (type C) or without purification of DNA by boiling (type D). The optimal efficiency of 2.016 was obtained on Roche LightCycler(®) 4.1. software for type C standards, whereas the lowest efficiency (1.682) corresponded to type D standards. Type C standards appeared to be more suitable for quantitative real-time PCR because of the use of defined populations for construction of standard curves. Overall, Fieller Confidence Interval algorithm may be improved for replicates having a low standard deviation in Cycle Threshold values such as found for type B and C standards. Stabilities of diluted PCR standards stored at -20°C were compared after 0, 7, 14 and 30 days and were lower for type A or C standards compared with type B standards. However, FACS generated standards may be useful for bacterial quantification in real-time PCR assays once optimal storage and temperature conditions are defined.

Responses of Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus to simulated food processing treatments, determined using fluorescence-activated cell sorting and plate counting.

Three common food pathogenic microorganisms were exposed to treatments simulating those used in food processing. Treated cell suspensions were then analyzed for reduction in growth by plate counting. Flow cytometry (FCM) and fluorescence-activated cell sorting (FACS) were carried out on treated cells stained for membrane integrity (Syto 9/propidium iodide) or the presence of membrane potential [DiOC2(3)]. For each microbial species, representative cells from various subpopulations detected by FCM were sorted onto selective and nonselective agar and evaluated for growth and recovery rates. In general, treatments giving rise to the highest reductions in counts also had the greatest effects on cell membrane integrity and membrane potential. Overall, treatments that impacted cell membrane permeability did not necessarily have a comparable effect on membrane potential. In addition, some bacterial species with extensively damaged membranes, as detected by FCM, appeared to be able to replicate and grow after sorting. Growth of sorted cells from various subpopulations was not always reflected in plate counts, and in some cases the staining protocol may have rendered cells unculturable. Optimized FCM protocols generated a greater insight into the extent of the heterogeneous bacterial population responses to food control measures than did plate counts. This study underlined the requirement to use FACS to relate various cytometric profiles generated by various staining protocols with the ability of cells to grow on microbial agar plates. Such information is a prerequisite for more-widespread adoption of FCM as a routine microbiological analytical technique.

Physiological response of Bacillus cereus vegetative cells to simulated food processing treatments.

Vegetative cells of the spore-former Bacillus cereus were exposed to a number of treatments commonly used in commercial food preparation or during equipment cleaning and decontamination. Treated suspensions were then analyzed for reductions (CFU per milliliter) by plate counting and changes in levels of ATP and ADP released from cells with a bioluminescence-based assay. With the use of flow cytometry (FCM), the physiological status of individual cells before and after exposure to treatments was determined by staining of control and treated cells with three pairs of physiological dyes (SYTO 9/propidium iodide, carboxyfluorescein diacetate/Hoechst 33342, and C12-resazurin/SYTOX Green). Good agreement was found between plate counting and FCM. In general, treatments giving rise to the highest count reductions also had the greatest effects on cell membrane permeability (measured with the use of propidium iodide or SYTOX Green), esterase activity (measured with carboxyfluorescein diacetate), or redox activity (C12-resazurin). FCM data demonstrated the extent of heterogeneity of vegetative cell responses to treatments in, for example, the treatment with 5% H2O2, which caused a 6-log reduction in which approximately 95% of the population was composed of membrane-damaged cells (as reflected by their permeability to SYTOX Green), whereas in treatment with 0.09% (wt/vol) potassium sorbate, which caused only a 1-log reduction, not more than 40% of cells were membrane damaged. The approaches described in this work can be applied to gain a greater understanding of bacterial responses to food control measures, generate more accurate inactivation models, or screen novel prospective food control measures.

Monitoring growth phase-related changes in phosphatidylcholine-specific phospholipase C production, adhesion properties and physiology of Bacillus cereus vegetative cells.

The physiological status and metabolic heterogeneity of Bacillus cereus cells within a culture during an 8-h batch fermentation process was measured using flow cytometry (FCM). Concurrently, production of the toxin, PC-PLC, and the extent of cell adhesion of live and dead cells were monitored using novel fluorescent assays. Flow cytometry analysis detected growth phase-related changes in the physiological profiles of cells over the course of the fermentation, with variation in the percentage of cells displaying membrane damage and intracellular esterase and redox activities. As the exponential phase proceeded, populations became more uniform in terms of protein content as measured using FCM in tandem with a cell tracking dye, with the majority of cells becoming membrane intact, esterase positive and redox active. PC-PLC activity appeared strongly related to cell density. Permeabilisation of cells was accompanied by a loss in adherent properties, while 25-100% of cells with intracellular esterase activity possessed adhesion properties. Cells in late exponential phase appeared to have reduced adherence properties compared to cells in early exponential or lag phase. As well as demonstrating the utility of FCM for measuring heterogeneity in terms of cell physiological status throughout the course of batch cultures, the methods utilised in this study could be used to relate processes such as toxin production or cell adhesion to cell physiological state.

Bacillus cereus endospores exhibit a heterogeneous response to heat treatment and low-temperature storage.

Bacillus cereus endospores were challenged by heat treatments simulating typical domestic/industrial cooking regimes and the resulting effects on germination, viability and sub-lethal heat damage determined using differential plate counting on a rich versus selective medium, flow cytometry (FCM), beta-D-glucuronidase (GUD) activity and OD(600) measurement. Additionally, these techniques were used to investigate the effect on endospores of storage in a non-nutrient medium at 4 degrees C for 1 month. Plate counting revealed that heating generated sub-populations of sub-lethally damaged endospores, with the more severe heat treatments generating larger proportions of sub-lethally damaged endospores. These findings were also reflected in FCM analyses, which detected large amounts of heterogeneity among the populations of heat-treated endospores and uncovered differences in the proportions of membrane-damaged endospores and those displaying esterase activity pre- and post-treatment. Plate count data suggested that both the control and heat-treated endospores lost viability during storage, with FCM data indicating that the proportion of membrane-damaged endospores increased and those displaying the esterase activity decreased. The FCM, GUD and OD(600) data suggested that germination rates decreased with the increasing severity of heat treatment. This study demonstrates that a combination of plate counting and FCM can be used to detect heterogeneity in the response of endospores to insults.

The use of flow cytometry to study the germination of Bacillus cereus endospores.

BACKGROUND: At present the study of endospore germination is conducted using microbiological methods which are slow and yield data based on the means of large heterogeneous populations. Flow cytometry (FCM) offers the potential to rapidly quantify and identify germination and outgrowth events for large numbers of individual endospores. METHODS: Standard methods were employed to arrest the germination of Bacillus cereus endospores at defined stages. Endospores were then stained with SYTO 9 alone or carboxyfluorescein diacetate (CFDA) together with Hoechst 33342 and analysed using FCM. Comparisons were made between FCM as a method to measure germination rate and standard microbiological techniques. RESULTS: Germinating endospores displayed increases in permeability to SYTO 9 and hydrolysis of CFDA compared with controls. Statistically significant correlations were found between the standard plate count method and both FCM methods for measuring the percentage of germinating and outgrowing endospores up to 75 min after addition of germinant. CONCLUSIONS: Using FCM, the percentage of germinating or outgrowing endospores at various time points during germination and/or outgrowth can be quantified. FCM with CFDA/Hoechst 33342 staining may be used to estimate overall germination rate, whereas FCM with SYTO 9 staining may be used to quantify ungerminated, germinating and outgrowing endospores.