Rapid endospore viability assay of Clostridium sporogenes spores

Wan-Wan Yang a, Adrian Ponce a,b,a California Institute of Technology 1200 E. California Blvd., Pasadena, CA, 91125, USAb Jet Propulsion Laboratory, 4800 Oak Grove Drive, Pasadena, CA, 91109, USA

a b s t r a c ta r t i c l e i n f o

Article history:Received 12 September 2008Received in revised form 20 April 2009Accepted 21 April 2009

Keywords:Clostridium sporogenesSporesTb-DPA luminescenceEndospore viability assay

A rapid Endospore Viability Assay (EVA), previously developed for Bacillus spores, was modied forenumeration of germinable Clostridium sporogenes spores. The EVA is based on the detection of dipicolinicacid (DPA), which is released during stage I germination and quantied by terbium (III) ion Tb-DPAluminescence. Germination of C. sporogenes spores in aqueous suspension was induced by L-alanine andNaHCO3 addition, and germinable endospore numbers were determined by reference to a standard curve.Determination of the fractions of germinable C. sporogenes spores by EVA and phase-contrast microscopyyielded comparable results of 54.0%2.9% and 59.3%2.6%, respectively, while only 32.3%5.3% of sporesproduced colonies on reinforced clostridial medium (RCM). Rates of germination were measured as afunction of temperature (30 C60 C) using EVA, yielding a linear relationship between the square root ofthe rate constant and inverse temperature.

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1. Introduction

Bacterial spores (endospores) are dormant cellular structures inthe life cycle of Bacillus and Clostridium (Sonenshein, 2000), and arearguably the hardiest life forms on Earth (Nicholson et al., 2000). C.botulinum and C. perfringens are food-poisoning agents (Peck et al.,2004), while psychrotrophic clostridia cause spoilage of chilledvacuum-packed meat, mesophilic clostridia introduce off-avordevelopment in cheese, and thermophilic clostridia cause spoilage ofcanned food (Peck et al., 2004). C. perfringens, C. difcile and C. tetaniare etiological agents in hospital acquired infection (Russell, 1990).Spores of sulte-reducing clostridia (SSRC) have been considered as anindicator for the presence of pathogenic microorganisms in drinkingwater by European Community legislators (Committee on Indicatorsfor Waterborne Pathogens, 2004). Due to their resistance to variousextreme conditions, Clostridium endospores are also employed asbiological indicators to monitor the effectiveness of various steriliza-tion processes (Johnston et al., 2005; Kanemitsu et al., 2005).

Currently, the standard method to quantify endospore viability isbased on culture dependent methods such as determination ofnumbers of colony forming units (CFU) from plate counts, whichrequires several days of incubation and tedious anaerobic techniquesfor Clostridium species. To facilitate quantication, Shafaat and Ponce(2006) developed an Endospore Viability Assay (EVA) for Bacillusspores based on the detection of a unique biomarker, dipicolinic acid(DPA). Despite exhibiting no detectable metabolic activity, spores areable to monitor the nutritional status of their environment and

germinate when conditions are favorable (Setlow, 2003). When spe-cic germinants are added to a spore suspension, spores proceedtowards germination and outgrowth via the following steps: 1) sporeactivation, 2) stage 1 germination, during which water partiallyrehydrates the spore core and DPA is released, 3) stage 2 germination,duringwhich cortex hydrolysis andmetabolic activity resumes, and 4)outgrowth (Setlow et al., 2003). DPA released from the spore coreduring stage 1 germination can be detected by adding TbCl3 to thespore suspension and measuring the resulting Tb-DPA luminescenceintensities (Supplemental Fig. 1A, B) (Shafaat, Ponce, 2006).

The DPA concentration can be correlated to the germinable sporepopulation, given an average DPA content per spore, and full DPArelease during germination (Setlow et al., 2003). This is the under-lying principle of EVA, in which the germinable spore population isdetermined by measuring the proportion of DPA released after ger-mination versus DPA released from the total spore population afterlysis by autoclaving.

We adapted EVA, previously used for Bacillus spores, to quantifytotal numbers of Clostridium spore and the fractions of these sporesthat could germinate. EVA was validated against traditional methodsof enumeration by phase contrast microscopy and plate counts andapplied to study the rate of germination of C. sporogenes spores as afunction of temperature.

2. Materials and methods

2.1. Materials

Deionized water (18.2 M/cm) was obtained from an ultraltersystem (Water Pro PS, LabConco, Kansas City, MO, USA). Terbium (III)

International Journal of Food Microbiology 133 (2009) 213216

Corresponding author. Jet Propulsion Laboratory, 4800 Oak Grove Drive, Pasadena,CA, 91109, USA. Tel.: +1 818 354 8196; fax: +1 818 393 4445.

E-mail address: ponce@caltech.edu (A. Ponce).

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chloride hexahydrate-(99.999%), dipicolinic acid (2,6-pyridinedicar-boxylic acid, DPA, 99%), L-alanine and L-lactate were purchased fromAldrich (Milwaukee, WI, USA). C. sporogenes, ATCC 7955 (AmericanType Culture Collection, Manassas, VA, USA) was obtained as a freeze-dried pellet.

2.2. Endospore production and purication

C. sporogenes was revived in approximately 5 mL of reinforcedclostridial medium (RCM; Becton Dickinson, Sparks, MD, USA) at 37 Cfor 3 days. The culture was transferred to fresh medium after devel-opment of visible turbidity. The production and purication of C.sporogenes spores was performed using a protocol developed spe-cically for Clostridium species (Yang et al., 2009). The spore sus-pension contained less than 1% vegetative cell material as determinedby phase contrast microscopy.

2.3. Endospore quantication by Tb-DPA luminescence

Tb-DPA luminescence excitation spectra (ex=250360 nm,em=544 nm) and emission spectra (ex=278 nm, em=450650 nm) were recorded with a uorimeter (Model FL-1089, JobinYvon, Edison, NJ, USA) consisting of a 500-W Xe-lamp for excitation,two double-monochromators set at 4-nm bandpass, and a Peletier-cooled photomultiplier tube (Model R928, Products for Research Inc,Danvers, MA, USA). A 500 nm cut-off lter (Omega Filters,Brattleboro, VT, USA) was placed at the entrance of the emissionmonochromator. Emission intensities recorded on different dayswere normalized by reference to the emission intensity of a Tb-DPAstandard solution.

To correlate luminescence intensity with spore concentration, 13triplicate suspensions of spores at numbers ranging from 106 to109 spores/ml were autoclaved at 136 C for 60 min to ensurecomplete release of DPA from spores. The luminescence intensityobtained from each spore concentration was plotted in a calibrationcurve (Fig. 1). The possibility of interference of germinants on theluminescence intensity was eliminated by adding to cali-bration suspensions the same concentrations of L-alanine (100 mM)and NaHCO3 (50 mM) as were used in validation experiments(Section 2.4.).

2.4. EVA determination of germinable and total spore concentrations

To determine the number of germinable spores in samples, germi-nation was induced in triplicate samples of suspensions containing

known total numbers of spores by adding 0.3 ml of 1 M L-alanine and0.15 ml of 1 M NaHCO3 to 2.55 ml of each spore suspension. The nalconcentrations of germinants were 100 mM for L-alanine and 50 mMfor NaHCO3. The supplemented suspensions were then incubated at37 C for 48 h to allow the germination process to go to completion.Following germination, 30 l of 1 mM TbCl3 was added to eachsuspension to obtain a nal concentration of 10 M TbCl3. Tb-DPAluminescence emission and excitation spectra of the mixtures wererecorded. The luminescence intensity was converted to the number ofgerminable spores/ml by reference to the calibration curve (Fig. 1).

To determine the total number of spores, each mixture used fordetermination of germinable spore numbers was sealed in a micro-wavable tube and autoclaved at 136 C for 60 min, to release DPA fromspores that had not germinated. After cooling to room temperature,the mixtures were transferred to acrylate cuvettes, and Tb-DPA lumi-nescence emission and excitation spectra were determined. The totalnumbers of spores/ml were obtained by reference to the calibrationcurve, and the proportion of spores that germinated was then calcu-lated as the ratio of germinated spores to total spores.

2.5. Phase contrast microscopic enumeration of total and germinablespores

Five l aliquots of spore suspensions were placed in a Petroff-Hausser counting chamber (Model 3900, Hausser Scientic, Horsham,PA, USA), and spores were observed at 400 magnication using aphase contrast microscope (Nikon Eclipse 80i, AG Heinze Co, LakeForest, CA,USA)ttedwith adigital camera (NikonDigital SightDS-5M).The smallest squares in the counting chamber are 0.05 mm0.05 mm,of which 80 were analyzed to obtain the average spore count persquare. To obtain statistically signicant counts, the spore concentra-tions above 107 spores/ml were used, which resulted in at least 10 cellsper sixteen squares. Spore suspensions with numbers in the range of51075108 spores/ml were prepared by appropriate dilution of thestock suspension.Non-germinated, intact spores are phase-bright,whilegerminated spores and vegetative cells are phase dark under phasecontrast illumination. Phase bright spores were counted before andafter germination. The total numbers of spores were determined fromcounts of phase bright spores before germination, while the numbers ofgerminable spores were determined from the differences in the num-bers of phase bright spores before and after germination.

2.6. Determination of CFU per milliliter of spore suspension

A heat shock treatment of 80 C for 15 min was applied to inac-tivate vegetative cells in spore suspensions for which the total number

Fig. 1. Tb-DPA luminescence intensity standard curve. Error bars represent standarddeviation.

Fig. 2. Germination time course of C. sporogenes spores, which shows Tb-DPAluminescence intensity as a function of time.

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of spores had been determined by phase contrast microscopy.Dilutions were prepared to obtain spore suspensions containing bet-ween 102 and 103 spores/ml. One hundred l aliquots from eachdilution were plated in triplicate on RCM supplemented with agarat 15 g/l, and incubated in a GasPak EZ Anaerobe pouch system(Becton Dickinson, Franklin Lakes, NJ, USA) at 37 C for 3 days.Colonies were counted and the mean number of CFU/ml in eachoriginal sample was calculated.

2.7. Temperature dependence of germination dynamics

The temperature dependence of spore germination dynamicswas determined by measuring the Tb-DPA luminescence intensityincrease as spores germinated (Fig. 2). Spore suspensions containing~107 spores/ml, supplemented with 100 mM L-alanine, 10 mMNaHCO3 and 10 M TbCl3, were incubated at 30 C, 37 C, 45 C and60 C. The germinants, L-alanine and NaHCO3, were added to the sporesuspensions at time zero. Tb-DPA luminescence emission spectrumintensity of each mixture was recorded at various intervals duringincubation until the luminescence intensity reached a plateau. Theplateau is reached when all germinable spores have germinated andcompletely released DPA on the experimental time scale and underthe germination conditions provided. Spore germination timecourseswere modeled using Gt=Ae rt (Chea et al., 2000), where Gt is the

luminescence intensity at time t, A is the plateau luminescenceintensity, and r is the germination rate constant. The best t to theexperimental data yielded the plateau intensity parameter (A) foreach incubation temperature. The timecourse data in terms of extentof germination was obtained by normalizing the luminescenceintensity timecourse data with respect to A.

3. Results and discussion

A linear correlation (R2=0.999) of Tb-DPA luminescence intensitywith spore concentration was determined for spore numbers in therange 106 to 109 spores/ml (Fig. 1). On the assumption that there is anaverage DPA content per spore, the average DPA content per C.sporogenes spore was calculated to be 3.7108 molecules per spore,which is comparable to the value of 1.72.2108 molecules per sporefor Bacillus spores (Hindle and Hall, 1999; Shafaat and Ponce, 2006).

During germination, DPA is released from endospores. However,C. sporogenes spores did not germinate upon the addition of only L-alanine, the most commonly used Bacillus spore germinant. Foroptimal germination of C. sporogenes spores, a combination of100 mM L-alanine and 50 mM NaHCO3 was required. Clostridiumspores also germinated upon the addition of L-lactate, but at a muchslower rate and to a lesser extent (Supplemental Fig. 2). Even underoptimized germination conditions, germination of the tested strain of

Fig. 3. (A) Correlation between phase-contrast enumeration and Tb-DPA luminescenceassay for quantication of total spore concentration. (B) Correlation between phasecontrast enumeration and Tb-DPA luminescence assay for quantication of germinablespores. The lines shown indicate a 1:1 correspondence.

Fig. 4. (A) The extent of germination over time at temperatures: 30 C (solid circles),37 C (crosses), 45 C (triangles) and 60 C (squares). Points on graph are experimentaldata, and lines indicate t to germination model, Gt=Ae rt. (B) Linear relationbetween 1/T andr, where T is the germination temperature and r is the correspondinggermination rate.

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C. sporogenes is approximately 10 times slower than that of Bacillusatrophaeus (Shafaat and Ponce, 2006).

On addition of L-alanine and NaHCO3 to a C. sporogenes sporesuspension, DPA was released from the core of the germinated sporesinto the bulk solution (Fig. 2). Tb-DPA luminescence intensity increasedover time and reached a plateau at approximately 60% sporesgerminated after 30 h. Phase contrast images after germination alsoshow approximately 60% spore germination. Supplemental Fig. 3 showsthe transition of germinating spores from phase bright to phase dark.

The EVA was validated against phase contrast microscopy fordetermination of both total and germinable endospore populationsover a range of 106107 spores/ml in spore concentration. There was aone-to-one correlation between phase-contrast and EVA determinationof the total and germinable spores/ml, indicated by a good t to a linewith a slope of unity (Fig. 3). This validates the EVA methodology withrespect to the traditional phase-contrast microscopy methodology.

The germination dynamics were accurately described by theequation for Gt for each temperature shown (Fig. 4A). From the bestts (R2N0.99 for each timecourse), we obtained germination rates (r),which increased by a factor of 5.6 as temperature (T) increased from30 C to 60 C. The temperature dependence data yielded a linearrelationship betweenr and 1/T (Fig. 4B, R2N0.99). At 4 C, the rate ofgermination occurred on the time scale of a month (data not shown).However, the germination timecourse data at 4 C did not t ourmodel (Gt) and a rate constant was not determined.

When we applied EVA, phase contrast enumeration, and CFUcounts on the same spore suspension to measure germinability andculturability, the percentage of spores that germinated after two daysof incubation was determined by EVA to be 59.3%2.6%, by phasecontrast enumeration to be 54.0%2.9%, and the percentage of sporesthat formed colonies by plate counts to be 32.3%5.3%. Not allgerminable spores yield vegetative cells capable of forming colonies,indicating that a subset is germinable-but-not-culturable. In addition,within the viable spore population, subsets may be viable-but-not-germinable (i.e., superdormant), or viable-but-not-culturable (VBNC),although these viable populations are not directly measurable.CFU measurements will most likely underestimate the number ofviable spores due to the existence of VBNC spores (Colwell andGrimes, 2000). In contrast, EVA will most likely overestimate thenumber of viable spores due to the existence of germinable-but-not-viable spores, although the possibility of superdormant endosporepopulations would reduce that overestimation.

Potential applications of EVA for Clostridium endospores includeevaluating the efcacy of sterilization regimens for food, wastewater,and medical equipment. To test a given sterilization regimen, weenvision that surrogate microorganisms (e.g., C. sporogenes spores)would be applied to the appropriate matrix (i.e., food, wastewater,medical equipment) at concentrations that are above the EVA limit

of detection. After the sterilization process is complete, the matrix isextracted, and the extract analyzed with the EVA. Ultimately, withimplementation of automated sample handling and analysis, we en-vision that the EVA will enable online monitoring of sterilizationprocesses for food processing facilities.

Acknowledgments

The authors would like to thank Dr. Stephanie A. Connon forediting and Dr. Christina N. Dock for insightful discussions. Theresearch described in this paper was carried out at the Jet PropulsionLaboratory, California Institute of Technology, under a contract withthe National Aeronautics and Space Administration.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.ijfoodmicro.2009.04.024.

References

Chea, F.P., Chen, Y., Montville, T.J., Schaffner, D.W., 2000. Modeling the germinationkinetics of Clostridium botulinum 56A spores as affected by temperature, pH, andsodium chloride. Journal of Food Protection 63, 10711079.

Colwell, R., Grimes, D. (Eds.), 2000. Non-culturable microorganisms in the environ-ment. InASM Press, Washington, D.C.

Committee on Indicators for Waterborne Pathogens, N.R.C., 2004. Indicators for Water-borne Pathogens. The National Academy Press, Washington, D.C.

Hindle, A.A., Hall, E.A.H., 1999. Dipicolinic acid (DPA) assay revisited and appraised forspore detection. Analyst 124, 15991604.

Johnston, M.D., Lawson, S., Otter, J.A., 2005. Evaluation of hydrogen peroxide vapour as amethod for the decontamination of surface contaminated with Clostridium botulinumspores. Journal of Microbiology Methods 60, 403411.

Kanemitsu, K., Imasaka, T., Ishikawa, S., Kunishima, H., Harigae, H., Ueno, K., Takemura, H.,Hirayama, Y., Kaku, M., 2005. A comparative study of ethylene oxide gas, hydrogenperoxide gas plasma, and low-temperature steam formaldehyde sterilization.Infection Control and Hospital Epidemiology 26, 486489.

Nicholson, W.L., Munakata, N., Horneck, G., Melosh, H.J., Setlow, P., 2000. Resistance ofBacillus endospores to extreme terrestrial and extraterrestrial environments.Microbiology and Molecular Biology Reviews 64, 548572.

Peck, M.W., Granum, P.E., Gould, G.W., Mainil, J.G., 2004. Foodborne clostridia andsporulation. Institute of Food Research, Norwich, UK.

Russell, A.D., 1990. Bacterial-spores and chemical sporicidal agents. Clinical Micro-biology Reviews 3, 99119.

Setlow, P., 2003. Spore germination. Current Opinion in Microbiology 6, 550556.Setlow, B., Cowan, A.E., Setlow, P., 2003. Germination of spores of Bacillus subtilis with

dedecylamine. Journal of Applied Microbiology 95, 637648.Shafaat, H.S., Ponce, A., 2006. Application of a rapid endospore viability assay for

monitoring UV inactivation and characterizing arctic ice cores. Applied andEnvironmental Microbiology 72, 68086814.

Sonenshein, A.L., 2000. Endospore-forming bacteria: an overview. In: Brun, Y.V.,Shimkets, L.J. (Eds.), Prokaryotic Development. InAmerican Society for Microbiol-ogy, Washington, D.C., pp. 133150.

Yang, W.-W., Crow-Willard, E., Ponce, A., 2009. Production and characterization of purespore suspensions of Clostridium sporogenes and C. hungatei. Applied Microbiology106, 2733.

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Rapid endospore viability assay of Clostridium sporogenes sporesIntroductionMaterials and methodsMaterialsEndospore production and purificationEndospore quantification by Tb-DPA luminescenceEVA determination of germinable and total spore concentrationsPhase contrast microscopic enumeration of total and germinable sporesDetermination of CFU per milliliter of spore suspensionTemperature dependence of germination dynamics

Results and discussionAcknowledgmentsSupplementary dataReferences