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

Gorsuch, John P.; Jones, Zachary; Saint, Daniel Le; Kitts, Christopher L.

doi:10.1016/j.mimet.2019.105682

Cited by 16 publications

(9 citation statements)

References 9 publications

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“…Samples of batch reactor flask medium (1.0mL) were collected from each replicate flask ( n = 3 per treatment) at 15-minute intervals over 105 min for APC assays, which were conducted as described in previous work (Gorsuch et al., 2019a) following a spread-plate technique. Culture medium was Tryptic Soy Agar (TSA) augmented with 0.075g.L bile salts (Millipore Sigma, Burlington, MA) and 0.025 g/L Congo red dye (Carolina Biological Supply, Burlington NC).…”

Section: Methodsmentioning

confidence: 99%

“…Global labeling and regulatory mandates require the accurate enumeration of Bacillus endospores in such products, and thus they are routinely subjected to enumeration assays. Growth-based plate counting methods such as the aerobic plate count (APC) are the industry standard for enumerating microbial products; however, even under optimal conditions these assays tend to underestimate microbial concentration (Davis, 2014; Sutton, 2012) and standard iterations are sometimes inappropriate for the enumeration of Bacillus -based products (Gorsuch et al., 2019a). Growth-independent enumeration methods such as quantitative polymerase chain reaction (qPCR), digital polymerase chain reaction (dPCR) and flow cytometry (FC) address many of the APC assay's limitations and thus represent attractive alternatives to plate counting (Davis, 2014; Gorsuch et al., 2019b).…”

Section: Introductionmentioning

confidence: 99%

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

Gorsuch¹,

Woodruff²

2019

Heliyon

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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.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Gorsuch¹,

Woodruff²

2019

Heliyon

Self Cite

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“…TSA agar (Tryptone soya agar; Oxoid, CM0131) were used for enumeration of Bacillus spp. (Gorsuch et al, 2019). One hundred µL of each sample dilution in saline was spread onto a TSA plate and aerobically incubated at 37 o C/48 h. Typical colonies of Bacillus bacteria were large, wrinkled, and saw-edged.…”

Section: Bacillus Sppmentioning

confidence: 99%

Quantities and antibiotic resistance of microorganisms in some microbial products for animals in Vietnam

Ho¹

2021

The J. Agric. Dev.

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The aims of the study were to look into the quantities of live beneficial microorganisms and antibiotic resistance of bacterial strains in several probiotic products used for food animals in the market. Ten probiotic products that claim to contain beneficial bacteria and fungi were examined. Eight products are said on the label to contain Lactobacillus spp., nine contain Bacillus spp., five contain yeasts and two have molds. The results showed that eight products did not have the microbial quantities or/and composition of microorganisms as saying on their labels. Of eight products which claim to contain Lactobacillus spp., the bacteria were isolated from only four, of which three had Lactobacillus counts at least ten-fold as low as the numbers on the labels. Spore-forming bacilli were isolated from all nine Bacillus-containing products. However, two products had the bacterial counts at least 10-fold as low as the numbers printed on the labels. Among five products stated to contain yeasts, the organisms were recovered from samples of only one. Seven Lactobacillus and fifteen Bacillus isolates from all samples that had bacterial growth were tested for their susceptibility against seven common antibiotics using Kirby-Bauer disk diffusion method. The results revealed that all the Lactobacillus isolates showed susceptibility to the tested antibiotics except kanamycin. All 15 Bacillus isolates were susceptible to ampicillin, kanamycin, and ciprofloxacin; five isolates were intermediately resistant to tetracycline; one isolates resisted erythromycin, and one isolates was resistant to vancomycin. The results of this study would provide information for farm practice in choosing antibiotics used together with antibiotics to maintain or/and restore the gut microflora after antibiotic treatment.

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“…Moreover, the growth of plated bacteria and their morphology, density and motility lead to an underestimation of CFU and consequently to a decreased bacterial cell count. CFU determination is a time‐consuming and elaborate method for spore detection due to sample preparation and incubation time [ 11 , 12 , 13 ]. Furthermore, endospores can also be marked by staining techniques such as Schaeffer‐Fulton to distinct between vegetative cells and endospores [ 14 , 15 ].…”

Section: Introductionmentioning

confidence: 99%

Facilitated endospore detection for Bacillus spp. through automated algorithm‐based image processing

Biermann

Niemeyer

Rösner

et al. 2021

Engineering in Life Sciences

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Bacillus spp. endospores are important dormant cell forms and are distributed widely in environmental samples. While these endospores can have important industrial value (e.g. use in animal feed as probiotics), they can also be pathogenic for humans and animals, emphasizing the need for effective endospore detection. Standard spore detection by colony forming units (CFU) is time-consuming, elaborate and prone to error. Manual spore detection by spore count in cell counting chambers via phase-contrast microscopy is less timeconsuming. However, it requires a trained person to conduct. Thus, the development of a facilitated spore detection tool is necessary. This work presents two alternative quantification methods: first, a colorimetric assay for detecting the biomarker dipicolinic acid (DPA) adapted to modern needs and applied for Bacillus spp. and second, a model-based automated spore detection algorithm for spore count in phase-contrast microscopic pictures. This automated spore count tool advances manual spore detection in cell counting chambers, and does not require human overview after sample preparation. In conclusion, this developed model detected various Bacillus spp. endospores with a correctness of 85-89%, and allows an automation and time-saving of Bacillus endospore detection. In the laboratory routine, endospore detection and counting was achieved within 5-10 min, compared to up to 48 h with conventional methods. The DPA-assay on the other hand enabled very accurate spore detection by simple colorimetric measurement and can thus be applied as a reference method.

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

Cited by 16 publications

References 9 publications

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

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

Quantities and antibiotic resistance of microorganisms in some microbial products for animals in Vietnam

Facilitated endospore detection for Bacillus spp. through automated algorithm‐based image processing

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