Biological Indicator

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Journal of Applied Microbiology 1998, 85 , 865874

Biological indicators for steam sterilization: characterizationof a rapid biological indicator utilizing Bacillus stearothermophilus spore-associated alpha-glucosidase

enzymeH. Albert 1 , D.J.G. Davies 1 , L.P. Woodson 2 and C.J. Soper 1 ~1 School of Pharmacy and Pharmacology, University of Bath, Bath, UK and 2 3M Health Care, Medical Products Technology Division, St Paul, MN, USA

6430/10/97: received 14 October 1997, revised 15 June 1998 and accepted 17 June 1998

H. ALBERT, D.J.G. DAVIES, L.P. WOODSON AND C.J. SOPER. 1998. The alpha-glucosidase enzymewas isolated from vegetative cells and spores of Bacillus stearothermophilus, ATCC 7953.Spore-associated enzyme had a molecular weight of approximately 92 700, a temperatureoptimum of 60 C, and a pH optimum of 7075. The enzyme in crude aqueous spore extract

was stable for 30 min up to a temperature of 65 C, above which the enzyme was rapidlydenatured. The optimal pH for stability of the enzyme was approximately 72. The alpha-glucosidase in crude vegetative cell extract had similar characteristics to the spore-associatedenzyme but its molecular weight was 86 700. The vegetative cell and spore-associated enzymeswere cross-reactive. The enzymes are postulated to derive from a single gene product, whichundergoes modication to produce the spore-associated form. The location of alpha-glucosidase in the spore coats (outside the spore protoplast) is consistent with the location of most enzymes involved in activation, germination and outgrowth.

INTRODUCTION

Sterilization is the act or process, physical or chemical, thatdestroys or eliminates all forms of life, especially micro-organisms. This term is absolute, i.e. a substance cannotbe partially sterile (Joslyn 1991). Due to the difculty inconrming sterility, a more practical denition of sterility hasbeen adopted, dening sterility as the process by which livingorganisms are removed or killed to an extent that they are nolonger detectable in standard culture media in which theyhave previously been found to proliferate (Joslyn 1991). Inpractice, assurance of a low probability of any living micro-organisms remaining is used as a measure of sterility. Sterilityof a particular item can only be conrmed by destructivetesting of the item, which is not practical for most purposes.

Sterilization monitoring improves the assurance that medi-cal devices have been adequately sterilized. Biological moni-toring is accepted as the most effective method. Biologicalindicators function by introducing highly resistant bacterial

Correspondence to: Lewis P. Woodson, 3M Health Care, Medical ProductsTechnology Division, Building 270-3N-02, St Paul, MN 55144-1000, USA(e-mail: [email protected]).~ Deceased.

1998 The Society for Applied Microbiology

spores into the sterilization cycle. If these spores aredestroyed, it is assumed that any contaminating organisms inthe load have also been killed, as these organisms have lowerresistance than the spores, and are present in lower numbers(Pug and Odlaug 1986). Daily biological monitoring (andmonitoring of each load containing implants) is recommendedby the Association of Operating Room Nurses (Associationof Operating Room Nurses 1994), the Joint Commission onAccreditation of HealthcareOrganizations (Joint Commissionon Accreditation of Healthcare Organizations 1994), theAmerican Hospital Association, and the Association for theAdvancement of Medical Instrumentation (Association for

the Advancement of Medical Instrumentation 1993). TheCenters for Disease Control recommend weekly biologicalmonitoring (plus each load containing implantable devices).

Biological indicators consist of an inoculated carrier con-tained within its primary pack ready for use, and providing adened resistance to the specied sterilization process. Aninoculated carrier is a carrier on which a dened numberof test organisms have been deposited (Association for theAdvancement of Medical Instrumentation; AAMI ST59).The test organism used for steam sterilization is B. ste-arothermophilus because of its high resistance and consistent


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866 H . ALBERT ET AL.

inactivation kinetics. The original format of biological indi-cators was inoculated paper strips inside envelopes, whichwere transferred to sterile culture medium following process-ing, and incubated for 7 d (Rutala etal . 1996). Sterilizationfailure was measured by turbidity of the growth medium.

Second generation biological indicators are self-containedsystems which comprise the spore strip and growth mediumrequired for recovery in a primary pack ready for use. Thesebiological indicators rely on a pH indicator to measure pro-duction of acid metabolites in the growth medium by out-growing spores and replicating cells. These systems removethe problem of contamination during manipulation enco-untered with the rst generation indicators, but 24168hreadout time is still required for detection of surviving spores(Rutala etal . 1996).

Recently, the third generation biological indicator,Attest TM 1291 Rapid Readout Biological Indicator (3M, StPaul, MN, USA), was developed for use in 132 C gravitysteam sterilization cycles (ash sterilization) in view of theneed for rapid results in emergency sterilization proceduresused in the operating room (Vesley etal . 1992). In 1996,another third generation biological indicator was developedfor use in 121 C gravity steam sterilization and 132 C vac-uum-assisted steam sterilization (Attest TM 1292 Rapid Read-out Biological Indicator; 3M) (Vesley etal . 1995). These thirdgeneration biological indicators have a dual readout system.The rapid portion detects active spore-associated a-glu-cosidase enzyme surviving the sterilization process within 1 3 h. The enzyme is a normal constituent of vegetative cells andspores of B. stearothermophilus. It is consistently detectable in

viable spores but is destroyed by steam sterilization just afterspore kill (Vesley etal . 1992, 1995). The survival of the spore-associated a-glucosidase enzyme following exposure to steamsterilization correlates well with spore survival. The secondreadout system comprises a pH indicator which detects acidmetabolites produced by outgrowing spores and replicatingcells, and gives conrmation of the rapid result within 24 168h (Vesley etal . 1992, 1995).

Alpha-glucosidases catalyse the hydrolysis of terminal,non-reducing a-glycosidic bonds in short-chain saccharides,produced by the action of other enzymes, e.g. amylases onstarch releasing D -glucose. Alpha-glucosidases from veg-etative cells of various bacteria have been isolated and char-acterized (Suzuki etal . 1976; Urlaub and Wober 1978; Suzukietal . 1979, 1982, 1984; Kelly etal . 1986 ). They are foundwidely in animals, plants and micro-organisms, and are usu-ally found in association with amylases (Sata etal . 1987) toeffect the complete degradation of starch and other macro-molecules (Kelly and Fogarty 1983). The substrate specicityof the enzyme is dependent on its source(Suzuki etal . 1976,1979, 1982; Kelly and Fogarty 1983; Suzuki etal . 1984). Thea-glucosidase of B. stearothermophilus ATCC 7953 is mostsimilar to the less common group of a-glucosidases which

1998 The Society for Applied Microbiology, Journal of Applied Microbiology 85 , 865874

have high activity towards p-nitrophenyl- a-D -glucoside(PNPG), and activity towards maltose and malto-oli-gosaccharides,but littleor no activity towards a-1,6glycosidicbonds, i.e. isomaltose and isomaltosaccharides (Kelly etal .1986). Microbial a-glucosidases can be intracellular, cell-

bound or extracellular enzymes (Urlaub and Wober 1978;Suzuki etal . 1979; Kelly etal . 1986).There is little information available regarding the presence

and potential role of bacterial spore-associated a-glucosidaseenzyme. This study was undertaken to characterize the a-glucosidase enzyme utilized as a rapid monitor of sterilizationefcacy. The extraction and characterization of a-glucosidasefrom vegetative cells and spores of B. stearothermophilusATCC 7953 are described. Factors affecting the activity of the enzyme, both during growth of vegetative cell culturesand spores and during spore germination, will be determined.The localization of the enzyme within the spore was deter-mined by a two-step electron microscopic immunolabellingmethod using anti- a-glucosidase monoclonal antibody andcolloidal gold-IgG complex, and a role of the enzyme in thefunction of the spore was postulated.

MATERIALS AND METHODS

Bacterial strains and growth conditions

Bacillus stearothermophilus was provided as spore strips (cul-ture number 213, June 1990) by 3M, St Paul, MN, USA,derived from ATCC 7953 culture. For culture maintenance,a spore strip was grown in tryptic soy broth (TSB) overnight

in a shaking incubator. This stock vegetative cell culture wasstored in 1 ml aliquots in liquid nitrogen. For the primaryinoculum, 1 ml of the stock vegetative cell culture was used.

Preparation of crude spore extract

A stationary phase culture was produced in TSB at 60 Cfrom the stock vegetative cell culture. This culture was usedto inoculate sporulation agar plates consisting of yeast extract4 g l

1 , nutrient broth 8 g l 1 , manganese chloride 01 g l

1

and agar 20 g l 1 , at a nal pH of 72 2 01. Sporulation agarplates were incubated, inverted, at 60 C for 72 h. Sporeswere harvested and washed using ice-cold deionized waterand stored at 4 C. Several batches of spore suspensionswere combined; the spores were suspended in 50 mmol l 1

phosphate buffer pH 74, and diluted to give a viable countof approximately 1 109 spores ml 1 . The spore suspensionwas divided into 20 ml volumes and sonicated on ice for5 min. The sonicate was centrifuged at 4000 rev min

1 for20 min. The supernatant uid was stored at 4 C. The sporepellet was suspended in fresh buffer and sonicated for a 1min period followed by centrifugation and resuspension infresh buffer until no discernible a-glucosidase was present in


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RAPID BIOLOGICAL INDICATOR FOR STEAM STERILIZATION 867

the supernatant uid (up to three times). The supernatantuids were combined and concentrated to a nal volume of 5 ml using ultraltration units 8010, 8050 and 8200 (AmiconDivision, W.R. Grace and Company, Danvers, MA, USA)and Diao ultraltration membranes (10000 molecular

weight retention). The concentrated spore extract was storedat 4 C. The crude vegetative cell extract was produced froma logarithmic phase culture in TSB at 60 C, using the son-ication method described above.

Chemicals and reagents

Microbiological reagents were obtained from Difco. Otherchemicals were of analytical reagent grade and supplied bySigma. Electron microscopic reagents were obtained fromAgar Scientic Ltd unless stated otherwise.

Alpha-glucosidase assayAlpha-glucosidase activity was measured using two methods.

Method 1. Alpha-glucosidase was determined by the spec-trophotometric measurement at 410 nm of the release of p-nitrophenol from the substrate p-nitrophenyl-alpha- D -glu-coside (PNPG). Assay mixtures consisted of 5 mmol l 1

PNPG with 100 ml of an appropriate dilution of the crudespore extract in a total volume of 800 ml in 50mmol l 1

phosphate/EDTA buffer pH 74. A Pye-Unicam UV/VISkinetics spectrophotometer was used for all absorbancemeasurements. The absorbance of the reaction mixture wasrecorded at 410nm at 15 s intervals in a temperature-con-trolled chamber at 60C. The linear reaction velocity (changein absorbance per minute) was calculated from the gradientof the linear portion of the reaction prole and used to deter-mine the a-glucosidase activity (mol PNP min 1 mg 1

protein).

Method 2. Alpha-glucosidase activity was determined by theuorimetric detection of 4-methylumbelliferone (4-MU)released from 4-methylumbelliferyl-alpha- D -glucoside (4-MUG). The buffer or culture medium was added to a mic-rotitre plate (Costar, Cambridge, MA, USA). The appro-priate volume of 4-MUG solution was added (01 g l 1 ). Thespore suspension or crude extract was added, and the reactionmixture was thoroughly mixed. The microtitre plate wasincubated for 30 min at 60 C. The uorescence ( l em 455 nm,l ex 360 nm) was measured immediately at room temperatureusing a plate-reading uorimeter (Fluoroskan II, Titertek,Northbrook, IL, USA).

Protein concentration was assayed by the method of Lowry(Lowry etal . 1951) using bovine serum albumin (BSA) asstandard.


Molecular weight determination

An SDS-PAGE minigel system (Pharmacia Biotech,Uppsala, Sweden) and a 125% SDS gel were used in con-junction with low range molecular weight markers (BioRad)to determine the molecular weights of the spore and veg-etative cell enzyme sub-units (Suzuki etal . 1976). A SephadexG-200 gel ltration column was used with molecular weightmarkers to separate the protein bands and assay the fractionsfor a-glucosidase activity. Gel ltration was also used todetermine the molecular weight of the intact enzymemolecule.

Immunolabelling

The samples of spore and vegetative cell extract were run onan SDS-PAGE gel, with molecular weight markers. Fol-lowing electrophoresis, the gel was rinsed in transfer buffer

for 1 h prior to being electrophoretically transferred to anitrocellulose membrane (BioRad) using a Mini TransblotCell (BioRad) at 100 V for 1 h. The nitrocellulose membranewas incubated in blocking buffer (01% Tween-20, 01%BSA in Tris-buffered saline) for 1 h at room temperature.The blocking buffer was removed and the membrane wasincubated in a 1 in 50 dilution of mouse antispore a-glu-cosidase monoclonal antibody in blocking buffer for 4 h atroom temperature. The primary antibody solution wasremoved and the membrane was thoroughly washed in block-ing buffer. A 1 in 50 dilution of the secondary goat antimousegold-conjugated antibody (British Biocell International, Car-

diff, UK) was added and incubated at room temperature for2 h. The membrane was rinsed with TBS. A red colourationof labelled bands was observed.

Properties of the alpha-glucosidase

The effect of pH on a-glucosidase activity was determinedby assaying the crude vegetative cell extract and crude sporeextract (375 10 4 mg protein) for a-glucosidase activity inSorensens phosphate buffers of pH between 50 and 82,with 5 mmol l 1 PNPG at 60C. The effect of pH on a-glucosidase activity was measured by diluting the crude veg-etative cell extract and crude spore extract (3 10 3 mgprotein) 1 in 10 in each of the So rensens phosphate buffers.The mixtures were incubated at 60 C for 30 min, then buff-ered PNPG at pH 74 was added to give a nal concentrationof 5mmol l 1 PNPG in the reaction mixture. The a-glu-cosidase activity was measured at 60 C.

The effect of temperature on a-glucosidase activity wasdetermined by measuring the a-glucosidase activity of crudevegetative cell extract and crude spore extract (375 10 4

mg protein) at a series of temperatures with 5 mmol l 1

PNPG at pH 74. Alpha-glucosidase stability was determined


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by incubating the crude vegetative cell extract and crudespore extract in phosphate/EDTA buffer pH 74 for 30 minat a series of temperatures. The a-glucosidase activity of theextract (375 10 4 mg protein) was determined at 62 C atpH 74 with 5mmol l 1 PNPG.

The substrate specicity of the spore-associated a-glu-cosidase was assessed using the following method. Solutions(10mmol l 1 ) of the following carbohydrates were prepared:maltose, maltotriose, maltotetraose, maltopentaose, mal-tohexaose, nigerose, trehalose, isomaltose, sucrose andglucose. A 100 ml aliquot of crude spore extract (003mg ml 1

protein) was incubated in a 10 mmol l 1 solution of eachcarbohydrate in 50 mmol l 1 phosphate buffer pH 74 for30min at 60C. A sample of each reaction mixture wassubjected to four to six ascents of ascending paper chro-matography (Whatman 54 lter paper) using n-buta-nol/pyridine/water (6:4:3) (Suzuki etal . 1984). Thechromatogram was air-dried. Reducing sugars were vis-ualized using a modied silver dip method (Suzuki etal .1976). Solution A wasprepared by diluting 10ml of saturatedsilver nitrate to 60 ml with water and then to 200ml withacetone. Solution B consisted of 20% of a solution of 10%sodium hydroxide and 80% methanol. Solution C was anaqueous solution of 05mol l 1 NaS 2 O 3 . The dried chro-matogram was dipped into solution A and allowed to air dry.It was then dipped into solution B until black spots appeared.The chromatogram was next washed with distilled water andplaced in solution C until background colouration disappeared.The chromatogram was rinsed with distilled water. The glu-cose solution was used as a standard to compare the position of

the reaction products on the chromatographic paper. Glucosewasproduced by reaction of a-glucosidase with various carbo-hydrates.

Kinetic parameters

The kinetic parameters, K m and V max , were determined usingthe direct linear plot (Eisenthal and Cornish-Bowden 1974;Cornish-Bowden and Eisnethal 1974). The a-glucosidaseactivity was determined by the PNPG assay using a series of substrate concentrations: 02mmol l 1 , 05mmol l 1 ,10 mmol l 1 , 15mmol l 1 , 20mmol l 1 , 25mmol l 1 ,30mmol l 1 and 50 mmol l 1 . The linear reaction velocitywas calculated from the gradient of the initial portion of thereaction prole for each substrate concentration. A Hanesplot (Fig. 6) was initially constructed to inspect the linearityof the data obtained visually. Pairs of ( v, s) values weregenerated in the usual manner. The data were analysed usingEnzpack 3 version 30 (published and distributed byBIOSOFT, Cambridge, UK). This software allowed the cal-culation of kinetic parameters by the Direct Linear Plotmethod and also the Hanes plot, Lineweaver-Burk plot andthe Eadie-Hoftsee plot.


Antibody production

Balb/c mice were immunized three to ve times with B. stearothermophilus ATCC 7953 spores by interperitonealinjection at 23week intervals, with a nal injection of 1 106 cells given intravenously 3 d prior to hybridizationof the mice spleen cells and Balb/c NS-1 myeloma cells in35% polyethylene glycol. The treated cells were allowed torecover for24 h in Heaps Dulbeccos Modied Eagle Medium(HDMEM) with 10% foetal calf serum. The cells werediluted to 75 105 ml 1 in hypoxyanthene aminopterin thy-midine (HAT) medium, and plated in microtitre plates, themedium replaced as required and observed for the appearanceof hybridoma growth. Supernatant uids from the resultinghybridoma cultures were screened for the production of sporea-glucosidase-specic antibody by Particle ConcentrationFluorescence Immunoassay (PCFIA) using the IDEXXScreen Machine (IDEXX Corporation, Portland, OR, USA).

IDEXX carboxylated particles were added to sodium phos-phate buffer pH 70, and incubated at 37 C for 30 min. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl (EDC)(05mg ml 1 ), a-glucosidase (1 mg ml 1 ) from B. ste-arothermophilus and 2% non-fat dry milk in borate bufferwere added. The beads were centrifuged and the pellet sus-pended in 005% sodium azide in sodium phosphate bufferpH 70. Binding of the cell culture supernatant uids to thea-glucosidase-coated beads was measured by the IDEXXScreen Machine. Balb/c mice primed with pristane andallowed to rest for 10 d were injected intraperitoneally withapproximately 2 106 cells of the cultures showing the high-est level of binding to spore a-glucosidase. The ascites uidwas collected and the monoclonal antibodies were puriedusing the Af-Gel Protein A MAPS 11 system (BioRad).The immunoglobulin fractions were eluted, pooled, dialysedovernight and stored in 002% sodium azide in PBS at 4 C.

Electron microscopy and immunolabelling

For preparation of spore sections for examination of ultra-structure (Fig. 1), spores were xed in 3% glutaraldehydeand 1% acrolein in 50 mmol l 1 phosphate buffer pH 74,then xed in 1% osmium tetroxide solution at room tem-perature for 1 h. Following washing in sterile distilled water,spores were serially dehydrated in 30%, 50%, 70%, 80%,90%, 95%, 100% acetone and 100% dry acetone. Sporeswere suspended in the following series of mixtures of dryacetone and medium hardness Transmit EM resin (TaabLaboratories Equipment Ltd, Reading, UK), respectively:3:1, 1:1 and 1:3, and stored in 100% resin for several days atroom temperature. The samples were polymerized in gelatincapsules at 70 C for 18 h prior to sectioning.

For immunoelectron microscopic localization, freshly pre-pared spores were xed using either freeze-drying or incu-


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12

12

000

Time (min)

A b s o r b a n c e a t

4 9 0 n m

5

R e l a t

i v e

f l u o r e s c e n c e

5000

0

10

08

06

04

02

6 7 8 91 2 3 4

4000

3000

2000

1000

10 11

Fig. 1 Production of a-glucosidase during vegetative cell growthof B. stearothermophilus, ATCC 7953 in TSB at 60 C. ( ),a-glucosidase (relative uorescence); ( ), absorbance at 490 nm

bation in 3% glutaraldehyde solution in phosphate bufferpH 74 at room temperature for 16 h. Spores were washedtwice in phosphate buffer pH 74. Glutaraldehyde-xedspores were serially dehydrated in 30%, 50%, 70%, 90%ethanol and 100% dry ethanol at room temperature. Sporeswere inltrated with LR White resin (London ResinCompany, London, UK) with 05% w/v benzoin methylether, in the following combinations of dry ethanol and resinmixture, respectively: 3:1, 1:1 and 1:3. Spores were thenincubated in several changes of 100% resin mixture andstored for 35 d at 4 C. Samples were polymerized by u.v.

light ( l 360nm) for 3 d in gelatin capsules at 4 C. Ultra-thin sections were cut with a glass knife on a OMU3 Reichertultramicrotome, and mounted on gilded nickel grids (EMLElectron Microscopy Laboratories Ltd, Stansted, UK).

Ultra-thin sections were incubated in blocking buffer (gel-atin 05%, bovine serum albumin 01%, Tween-20 01% inTris buffer pH 82) for 5 min at room temperature. Thegrids were transferred to appropriate dilutions of monoclonalantibody in blocking buffer and incubated for 2 h. The gridswere washed in blocking buffer for 30 min, then incubated ina 1 in 10 dilution of gold-conjugated secondary antibody(Super EM Grade gold conjugate goat antimouse IgG, 15 nmgold; British Biocell International, Cardiff, UK) in blockingbuffer for 30 min and washed in sterile distilled water. Sec-tions were viewed using a Jeol JM 1200EX electron micro-scope (Tokyo, Japan) at 80 kV.

RESULTS

After an initial lag phase at the start of vegetative cell growth,the a-glucosidase activity increased during logarithmicgrowth of the culture (Fig. 1). Activity reached a plateau atthe onset of stationary phase (45 h) and the a-glucosidase


activity rapidly reduced after 8 h growth under these exper-imental conditions.

The optimum pH for enzyme activity was approximatelypH 78 (Fig.2). The optimum pH for enzyme stability wasapproximately 735 (Fig. 3). The activity of the enzyme

increased rapidly from 20 C, reaching an optimum activityat approximately 63 C in the crude extracts (Fig. 4). The a-glucosidase activity dropped sharply at temperatures between63 C and 77 C. The a-glucosidase was stable up to approxi-mately65 C, above which its stability rapidly decreased(Fig.5). This high thermostability of the enzyme in liquidextract reects theexpected increase in stabilityof the enzymein anhydrous form, and its use as a monitor of steam ster-ilization. Characteristics of the spore-associated and veg-

85

0016

000055

pH

R e a c t

i o n v e

l o c i

t y

( m o

l P N P m

i n 1

m g

1 p

r o t e i n )

0010

80

00140012

0008

0006

0004

0002

60 65 70 75

Fig. 2 Effect of pH on the reaction velocity of a-glucosidase.Reaction was carried out using crude cell or spore extract of Bacillus stearothermophilus, ATCC 7953 (375 10 4 mg ml 1

protein), 5 mmol l 1 PNPG at 60 C. ( ), Vegetative cell enzyme;( ), spore enzyme

84

0014

000064

pH

R

e a c t

i o n v e

l o c i

t y

( m o

l P N

P m

i n 1

m g

1 p

r o t e i n )

0010

74

0012

0008

0006

0004

0002

66 68 70 72 76 78 80 82

Fig. 3 Effect of pH on the stability of a-glucosidase. Reactionvelocity (at pH 74, 60 C, 5 mmol l 1 PNPG) was determined afterincubation for 30 min at 60 C at a series of pH values. Crudevegetative cell and spore extracts of Bacillus stearothermophilus,ATCC 7953 were used (375 10 4 mg ml 1 protein). ( ),Spore enzyme; ( ), vegetative cell enzyme


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80

0020

000020

Temperature ( C)

R e a c t

i o n v e

l o c i

t y

( m o

l P N P m

i n 1

m g

1 p

r o t e i n )

0015

70

0010

0005

30 40 50 60

Fig. 4 Effect of temperature on the reaction velocity of a-glucosidase. Reaction was carried out with crude vegetativecell or spore extract of Bacillus stearothermophilus, ATCC 7953(375 10 4 mg ml 1 protein), 5 mmol l 1 PNPG at pH 74. ( ),Vegetative cell enzyme; ( ), spore enzyme

80

0025

000020

Temperature ( C)

R e a c t

i o n v e

l o c i

t y

( m o

l P N P m

i n 1

m g

1 p

r o t e i n )

0015

70

0010

0005

30 40 50 60

0020

Fig. 5 Effect of temperature on stability of a-glucosidase.Reaction velocity (pH 74, 60 C, 5 mmol l 1 PNPG) wasdetermined after incubation for 30 min at a series of temperatures. Crude extracts of vegetative cells and sporesof Bacillus stearothermophilus, ATCC 7953 (375 10 4 mg ml 1

protein) were used. ( ), Vegetative cell enzyme; ( ), spore enzyme

etative cell forms were very similar, with the spore-associatedenzyme generally having a broader optimum range. The kin-etic parameters of the cell and spore-associated forms weresimilar, K m values 1405 mmol l 1 and 144 mmol l 1 PNPG,respectively (Table 1).

Glucose was produced by reaction of spore-associated a-glucosidase with maltose and the malto-oligosaccharides ( a-1,4). A small amount of glucose was detected following 30 minincubation at 60 C of a-glucosidase with nigerose ( a-1,3)and trehalose ( a-1,1). However, no glucose was formed byincubation with sucrose ( a-1,2) or isomaltose ( a-1,6). Themolecular weight of the spore a-glucosidase was 92 700, andthat of the vegetative cell enzyme was 86 700. The properties


2

5

12

0s

s / v

6

8

4

1 3 4

10

2

slope = 1/V max

Km /Vmax

Fig. 6 Hanes plot for determination of kinetic parameters, K mand V max . Reaction was carried out with crude vegetative cell extractin 5 mmol l 1 PNPG in phosphate/EDTA buffer pH 74 at60C

Table 1 Kinetic parameters, K m and Vmax , for vegetative cell andspore-associated a-glucosidase in crude extract (375 10 4 mgml 1 protein) in PNPG at pH 74, 60C. Parameters are statedas the mean 2 S.D.

Vmax (mol PNP min 1

K m (mmol l 1 ) mg 1 protein)

Vegetative cell 1405 2 0095 001119 2 000003Spore 144 2 009 000046 2 000004

of the a-glucosidase enzyme from vegetative cells and sporesof B. stearothermophilus ATCC 7953 were compared witha-glucosidases from other Bacillus species (Table 2). Theseenzymes vary in size, optimum conditions for activity andcatalytic properties.

Figure 7 shows the correlation between a-glucosidaseactivity (measured by uorescence after 1 h incubation) and24 h growth readout of Attest TM 1291 Rapid Readout Bio-logical Indicators following exposure to 132 C gravity ster-ilization. Very good correlation existed between 1 huorescence and 24 h growth readout results. The enzymeremained viable longer than the outgrowth of the spores,giving a margin of safety for the use of the uorescencereadout result.

Figure 8 shows the effect of the length of sporulation onthe activity of a-glucosidase during the germination and out-growth of spores with varying lengths of sporulation. Ger-mination of the spore suspension wasmeasured by a reductionin absorbance at 490nm. Samples of the germinating sporeswere assayed for a-glucosidase activity by uorimetric detec-tion. There was no increase in a-glucosidase activity above


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Table 2 Properties of several a-glucosidases from the genus Bacillus

Temperatureoptimum pH

Organism MW (C) optimum Specicity of bond cleavage Ref.

Bacillus stearothermophilus ATCC 7953 86 700 (c) 63 77 92 7 00 (s) 5663 7077 a-1,4 a-1,3, a-1,1. No a-1,2 or a-1,6

Bacillus stearothermophilus ATCC 12016 47 000 70 63 a-1,4. No a-1.6 Hunter 1993Bacillus thermoglucosidius KP1006 60 000 60 68 a-1,4 a-1,6 Cornish-Bowden

and Eisenthal1974

Bacillus cereus ATCC 7064 60 000 41 67 exo a-1,6 Eisenthal andCornish-Bowden1974

Bacillus licheniformis 66 000 40 60 a-1,4 a-1,6 Kelly and Fogarty1983

Bacillus amyloliquefaciens 27 000 39 68 a-1,4 a-1,2 a1,6 Kelly et al . 1986(c), Cellular form; (s), spore form.Data from this study. Data not obtained.

100

0

Exposure time (min)

P e r c e n

t a g e p o s i

t i v e r e s u

l t s

80

60

40

20

1 15 175 2 225 25 3

Fig. 7 Correlation of spore and a-glucosidase survival followingexposure of 3M Attest TM 1291 Rapid Readout BiologicalIndicators to 132 C gravity steam sterilization. ( ), 60 minuorescent result; ( ), 24 h growth result (colour change)

the basal level during the rst 2 h of incubation in any of thespore crops. After this period, the spores started to produceincreasing amounts of enzyme. The highest levels of a-glu-cosidase were detected in the spores with a 1 d sporulationperiod. The activity of a-glucosidase decreased as the lengthof sporulation of the spore crops increased.

The general appearance of sections of B. stearothermophilusspores was highly variable due to inherent heterogeneity of spore populations and alignment of spores in the resin duringsectioning. Figure 9 shows a typical electron micrograph of the ultrastructure of a dormant B. stearothermophilus spore.


6

700

00

Time (h)

F l u

o r e s c e n c e u n

i t s

1

600

200

100

2 3 4 5

300

400

500

Fig. 8 Effect of length of sporulation period on the a-glucosidaseactivity of spore suspensions during germination in TSB at60 C. Length of sporulation period: , 1d; , 2d; R , 3d; T ,4 d; E , 5 d

Sections of freeze-dried or glutaraldehyde-xed spores werereacted with anti- a-glucosidase monoclonal antibodies andlabelled with gold-conjugated secondary antibodies. The col-loidal gold particles were localized almost exclusively in theouter coats of dormant spores. Very few gold particles wereassociated with spores that had undergone control treatments.Similar results were obtained with freeze drying or glu-taraldehyde xation. As the conditions used for glu-taraldehyde xation were shown to reduce the activity of a-glucosidase signicantly(unpublished data), it is probable thatthe antibody binds at a site distinct from the active site.


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Fig. 9 A typical electron micrograph of the ultrastructure of adormant Bacillus stearothermophilus ATCC 7953 spore

A high level of background labelling was initially enco-untered when standard blocking buffer was used (Naish 1989;

Hunter 1993), due to binding of the gold particles to sulphurpresent in cysteine-rich proteins of the spore coats. Thisbackground labelling was particularly troublesome in thissituation as the spore coat was a likely site of a-glucosidaseactivity. Addition of 05% gelatin to the blocking buffereliminated this source of background labelling due to com-petitive binding of the gold and gelatin.

DISCUSSION

The location of the a-glucosidase outside the spore protoplastis in common with most enzymes involved in activation,germination and outgrowth. Immunoelectron microscopiclocalization of the Ger A proteins (Ger AA, Ger AB andGer AC), putative receptor proteins for the spore germinant,L -alanine, found these proteins to be located on the innersurface of cortex-less spore coat fragments (Sakae etal . 1995).Ger AB protein was also localized just inside the spore coatlayer of B. subtilis spores using transmission electronmicroscopy (Yasuda etal . 1996). Although bacterial sporesare metabolically dormant, they retain an alert sensory mech-anism which allows them to detect germinants in their exter-nal environment and trigger germination. The systems


Fig. 10 Immunoelectron microscopic localization of a-glucosidase enzyme in dormant spores of Bacillus stearothermophilusATCC 7953 xed in 3% glutaraldehyde in phosphate buffer pH74. Ultrathin sections were labelled with normal mouseserum and colloidal gold (15 nm)-IgG complex. Bar, 200 nm

responsible for germination triggering must escape the mech-anisms of stabilization which result in spore dormancy, at thesame time retaining the resistance properties of the wholespore (Foster and Johnstone 1989). The location of the a-glucosidase in the outer spore coats would also correlate withhigh inherent thermostability of the enzyme, as it would notbe inuenced by stabilizing factors present in the spore core.

The properties of the spore-associated and vegetative cellenzymes were very similar and the antispore a-glucosidaseantibody bound to both the spore-associated and vegetativeforms of the enzyme. The molecular weight difference isequivalent to approximately 4550 amino acid residues. It islikely that post-translation modication of the vegetative cellenzyme leads to the increased size of the spore form. Thismodication is presumed to allow specic targeting of the a-glucosidase to its site within the spore.

The a-glucosidase proved to be a useful predictor of sporesurvival as it is consistently present in viable spores andvegetative cells, and it survives just longer than the sporefollowing moist heat sterilization (Vesley etal . 1992). Itslocation in the spore coat suggests that it is necessary in theearly stages of germination or outgrowth, prior to new enzyme


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Fig. 11 Immunoelectron microscopic localization of a-glucosidase enzyme in dormant spores of Bacillus stearothermophilusATCC 7953. Ultrathin sections were labelled with anti- a-glucosidase monoclonal antibodies and colloidal gold (15 nm)-IgG complex. (a) Spores xed with 3% glutaraldehyde inphosphate buffer pH 74 for 16 h at 4 C. (b) Spores xed byfreeze drying in nitrogen slush. Bars, 200nm


synthesis. Enzymes not required during germination andoutgrowth of spores can be synthesized at a later stage duringvegetative growth.

Synthesis of a-glucosidase, L -alanine dehydrogenase andalkaline phosphatase during spore outgrowth were studied in

B. cereus strain T spores (Kobayashi etal . 1965). Synthesisof these enzymes began a few minutes after the start of germination; DNA synthesis began 80120min later. Thedormant spore is essentially lacking in stable, functionalmRNA, and its conversion to vegetative growth is dependenton mRNA transcription. These data further suggest that thea-glucosidase is required for spore outgrowth and normalvegetative cell function.

ACKNOWLEDGEMENTS

The authors would like to express their sincere gratitude toUrsula Potter, Department of Electron Optics, University of Bath, for her invaluable assistance. They also thank ChrisBinsfeld for monoclonal antibody production. The electronmicroscope was funded by the Science and EngineeringResearch Council. H. Albert was in receipt of a PhD stud-entship, funded by 3M Healthcare, St Paul, MN, USA.

REFERENCES

American Society for Healthcare Central Service Personnel of theAmerican Hospital Association (1989) Recommended Practices for Central Service, Sterilization . Chicago, IL: American HospitalAssociation.

Association for the Advancement of Medical Instrumentation (1993)Good Hospital Practice: Steam Sterilization and Sterility Assurance .Arlington, VA: Association for the Advancement of MedicalInstrumentation.

Association of Operating Room Nurses (1994) Proposed rec-ommended practices for sterilization in the practice setting . Association of Operating Room Nurses Journal 52, 109120.

Cornish-Bowden, A. and Eisenthal, R. (1974) Statistical con-siderations in the estimation of enzyme kinetic parameters by theDirect Linear Plot and other methods. Journal of Biochemistry139, 721730.

Eisenthal, R. and Cornish-Bowden, A. (1974) The Direct LinearPlot. Journal of Biochemistry 139, 715720.

Foster, S.J. and Johnstone, K. (1989) The trigger mechanism of bacterial spore germination. In Regulation of Prokaryotic Devel-opment ed. Smith, I., Slepecky, R.A and Setlow, P. pp. 89108.New York: Plenum Publishing Corporation.

Hunter, E. (1993) Practical Electron Microscopy. A Beginners Illus-trated Guide 2nd edn. Cambridge: Cambridge University Press.

Joint Commission on Accreditation of Healthcare Organizations(1994) Accreditation Manual for Hospitals. Oakbrook Terrace, IL: Joint Commission on Accreditation of Healthcare Organizations.

Joslyn, L.J. (1991) Sterilization by heat. In Disinfection, Sterilization,and Preservation 4th edn, ed. Block, S.S. pp. 495526. Phi-ladelphia: Lea and Febiger.


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