Meat extract casein peptone agar - A novel culture medium for the enumeration of Bacillus endospores in commercial products.

Here we propose a novel culture medium, Meat Extract Casein Peptone (MECP) agar, to support the enumeration of Bacillus endospores in commercial products. The formulation is the result of screening eight different veterinary, pharmaceutical, and industrial grade peptones for the ability to support the formation of small, well-defined Bacillus colonies on solid culture medium. The impact of agar purity, agar formulation rate, and metal cation additives were examined in prototype medium batches prepared from preferred peptone inputs. A customized plate counting assay based on the resultant MECP agar formulation was compared with standardized pour-plate and spread-plate assays (ISO 4833) and flow cytometry for the ability to accurately enumerate five Bacillus-based biostimulants and biofertilizers. Estimations of Bacillus endospore concentration generated by the customized spread-plate assay were significantly higher than those produced by ISO 4833 pour-plate and spread-plate assays for four out of the five tested products and were in better agreement with flow cytometry values; however, flow cytometry values were numerically higher than values returned by both plating methods. Both flow cytometry and plating assays based on MECP or similar culture media represent potential candidates for standardization and validation through organizations such as ISO and AOAC International for the enumeration of Bacillus-based products.

Incubation temperature and culture medium formulation impact the accuracy of pour-plate techniques for the enumeration of industrial Bacillus assemblages.

Aerobic plate counting assays based on the pour-plate technique are frequently used to enumerate microbial products; however, colony swarming and merging at the agar surface can reduce the accuracy of these assays. Some plating methods mitigate this risk through the inclusion of strategies including agar overlays; however, these interventions may be inadequate to mitigate swarming and merging of certain Bacillus colonies. In the present study, we assessed the accuracy of several pour-plate techniques for the enumeration of a mixed-species Bacillus assemblage. Tested modifications included a customized culture medium formulation, agar overlays, decreased incubation times and increased incubation temperature. Methods which produced countable plates were assessed for agreement with a Bacillus-specific plate counting assay and with total cell counts rendered by flow cytometry. While all tested pour-plate methods underestimated Bacillus endospore concentrations relative to flow cytometry and customized spread-plating, our results suggest that increasing incubation temperature and the inclusion of bile salts into culture medium formulations can improve the accuracy of pour-plate techniques when used to enumerate Bacillus assemblages by decreasing the incidence of spreading colonies. As Bacillus endospore preparations become more ubiquitous in the market, familiar enumeration methods such as the pour-plate technique may require methodological modifications to ensure that the cGMP compliance of Bacillus-based microbial products is assessed accurately.

Customized antimicrobial efficacy tests offer superior evaluation of growth inhibitor efficacy for liquid microbial products.

Antimicrobial effectiveness tests are common methods used to assess the risk of microbial contamination in pharmaceuticals and cosmetics. These assays may be inappropriate for endospore-based microbial products which often carry a similar - if not greater - risk of microbial contamination. In the present study, we compared the antimicrobial efficacy assessment provided by United States Pharmacopeia Chapter <51> Antimicrobial Effectiveness Testing with a modified test which utilized a customized bacterial challenge. The customized challenge inoculum comprised an assemblage of 12 bacterial strains (both pathogens and spoilage organisms) isolated from the product's end-use geography. Results suggest that some microbial inhibitor systems which pass industry standard antimicrobial effectiveness tests may fail when challenged with a customized bacterial assemblage. In order to provide the best possible assessment of microbial inhibitor systems for liquid Bacillus products, we suggest that new antimicrobial effectiveness tests be developed for this product class which include the addition of field-relevant contaminants in addition to the industry standard pathogen challenge.

Culture medium brand choice impacts colony swarming behavior among industrial Bacillus isolates and the accuracy of aerobic plate counts.

Aerobic plate count assays are an industry standard method for the enumeration of microbial products. Colony swarming among industrial Bacillus isolates on solid medium can impact a counting technician's interpretation of colony count, promote inter-technician variance and reduce the agreement of plate counts with growth-independent enumeration methods. In the present study, we examined swarming behavior among four industrial Bacillus species as a function of culture medium brand choice. Colony diameter for three Bacillus species was found to be influenced by culture medium brand, as was colony count interpretation among three out of four plate counting technicians. Estimations of Bacillus endospore concentration were likewise influenced by culture medium brand, leading to an increased incidence of QC failure for replicate samples of a Bacillus-based microbial product as a function of brand choice. Results suggest that culture medium brand choice may be an additional source of variance when plate counting is used for the enumeration of Bacillus-based microbial products. We recommend that plating medium brand availability in testing laboratories be considered by microbial product manufacturers when considering sources of variance in customer and regulatory laboratories.

Nutrient germination improves DNA recovery from industrial Bacillus subtilis endospores during qPCR enumeration assays.

Growth-independent microbial enumeration methods such as quantitative PCR require the efficient extraction of genomic DNA from targeted cells. Bacillus endospores are popular inclusions in commercial products due to their hardiness and metabolic dormancy; however, this hardiness is known to render Bacillus endospores resistant to traditional DNA isolation techniques. Metagenomic studies have sought to address this resistance through nutrient-based germination of bacterial endospores in environmental samples. In the present study, we sought to apply this technique to the enumeration of microbial products using an industrial strain of Bacillus subtilis as a model organism. Germination was induced through incubation of axenic spore suspensions in an AGFK-based rich medium. Total spore count, dipicolinic acid release and OD600 absorbance were monitored over time to track the progression of spore populations through the stages of germination and outgrowth. Aerobic plate counts and flow cytometry were used to monitor cell populations for proliferation during the incubation period. Finally, quantitative PCR with taxon-specific primers was used to examine DNA recovery as a function of time. Results show that customized germination protocols, once appropriately validated for the species and product matrix under consideration, can result in more efficient DNA extraction and thus lower limits of detection for qPCR assays targeting industrial Bacillus endospores in microbial products.

Enumeration of industrial Bacillus assemblages in commercial products with customized plate-counting assays.

The aerobic plate count assay remains among the most widespread methods for enumerating industrial Bacillus assemblages, as growth-independent methods are either cost prohibitive or unavailable in some areas. However, the standard plating assays used to verify the CFU count of Bacillus-based products are not tailored to Bacillus species and thus may not produce the most accurate possible estimations. Standard plating assays assume that established limits of quantification are applicable to Bacillus species whose colonies swarm on solid media, and that colonies of each species in a mixed-species assemblage form independently of one another on agar plates. In the present study, we examined the upper limit of quantification for an assemblage of swarming industrial Bacillus isolates by comparing plate count on medium with and without a swarming inhibitor with direct counts for spore suspensions of increasing endospore concentration. Additionally, we examined the impact of assemblage species composition on the evenness of colony distribution across replicate plates for four industrial Bacillus isolates. We compared the observed distribution of colonies across replicate plates to the expected Poisson distribution for axenic endospore suspensions, for a 3-species assemblage and a 4-species assemblage, respectively. Results suggest that customized plating assays may be more appropriate than standard protocols for the enumeration of Bacillus-based products, and that interactions between colonies on solid media should be considered when interpreting plating data for mixed-species Bacillus assemblages.

A comparison of methods for enumerating bacteria in direct fed microbials for animal feed.

Aerobic plate counts are the standard enumeration method for probiotic-containing products. This counting method is limited by the ability of many cells to enter a viable but non-culturable (VBNC) state upon exposure to stressful conditions like dehydration and heating commonly used in probiotic product preparation. Alternative enumeration methods are available including flow cytometry (FC) which counts total live/dead cells by assessing cellular integrity and/or metabolic activity, and quantitative polymerase chain reaction (qPCR) in which enumeration is correlated with the quantity of a nucleic acid target. These three methods were compared for enumerating three lactic acid bacteria (LAB): Pediococcus acidilactici, Pediococcus pentosaceus, and Lactobacillus plantarum, and a Bacillus subtilis related strain in twenty samples of a mixed probiotic product ranging in age from one to 825 days post-production. Flow cytometry and qPCR enumerations were similar and much higher compared to plate counts at later storage times, suggesting that some strains in the population were entering the VBNC state and were only countable by FC and qPCR. We propose the use of FC and/or qPCR as an alternative to plate counts for more accurate enumeration of bacteria in probiotic products.