Vol. 54, No. 10

Outermost-Cell-Surface Changes in an Encapsulated Strain ofStaphylococcus aureus after Preservation by Freeze-Drying

TOSHICHIKA OHTOMO,* TETSUO YAMADA, AND KOSAKU YOSHIDA

The Department of Microbiology, St. Marianna University School of Medicine, 2-16-1, Sugao, Miyamaye-ku,Kawasaki 213, Japan

Received 18 February 1988/Accepted 7 July 1988

The effects of drying time during freeze-drying on the outermost cell surface of an encapsulated strain ofStaphylococcus aureus S-7 (Smith, diffuse) were investigated, with special attention paid to capsule and slimeproduction. To quantify capsule and slime production, capsule antigen production and cellular characteristicssuch as growth type in serum-soft agar, cell volume index, and clumping factor reaction were examined. Afterfreeze-drying the colonial morphology of strain S-7 was altered from a diffuse to a compact type in serum-softagar. In accordance with these changes, the titer of the clumping factor reaction increased while the cell volumeindex, capsule and slime production, and capsule antigen production were markedly decreased in parallel withthe period of freeze-drying. The ability of the strain to adhere to collagen, fibrinogen, and soybean lectin wasalso compared before and after freeze-drying. Fibrinogen levels slightly increased when 10% skim milk and 2%honey were used as cryoprotective agents and showed a remarkable increase when 0.05 M phosphate buffer wasused as a control. Also, the ability of strain S-7 to adhere to soybean lectin declined, whereas no changes wereobserved for collagen under any conditions. Strain S-7 was phage nontypable before freeze-drying but thenumber of typable cells increased after freeze-drying; phage-typable cells reacted to phage 52 alone after 5 hof freeze-drying, but additional cells also proved to be phage typable to phage 42E after 10 h. Electronmicrographs indicated that strain S-7, an encapsulated strain, was converted to an unencapsulated state afterfreeze-drying. Results of our study indicate that the freeze-drying process inhibits capsule and slime productionin S. aureus, which consequently brings about changes in the outermost cell surface.

In many studies the effects of freezing and freeze-dryingon the cell surfaces of various microorganisms have beenexamined; however, most of them were concerned withinjury to the cell membrane (2, 6) or cell wall (8, 24, 25), andthere have been few reports on the effects of drying timeduring freeze-drying on capsule or slime biosynthesis. At-tention has recently been focused on material outside the cellwall, collectively known as glycocalyces (3), which includethe capsule and slime. Other studies have revealed how suchmaterials interact with the host cell (4, 7).

Encapsulated strains of Staphylococcus aureus have beenshown to be important for virulence; the slime or capsule ofthe strains has been studied in terms of infection, immunity(14, 21, 34-36, 39, 42, 43), and adherence (15, 36). However,surface materials are often lost in the process of preserva-tion, as with other encapsulated bacteria (26). We havepreviously reported on changes in biological activity andserological properties in encapsulated strains of S. aureusafter 10 years of freeze-drying preservation (41) and thepossible loss of capsules or slime (17). However, changes inthe outermost-cell-surface properties of microbes caused byfreeze-drying have not been elucidated previously. In thisreport we discuss changes which occurred on the outermostcell surface of an encapsulated strain of S. aureus afterpreservation by freeze-drying, with particular considerationgiven to the biological properties, capsule and slime produc-tion (CSP), capsule antigen production, adherence ability,and phage typability.

MATERIALS AND METHODSMicroorganism. An encapsulated strain of S. aureus, S-7

(Smith diffuse), was selected for this study (18, 20). Strain

* Corresponding author.

S-7 was positive for coagulase, phosphatase, DNase, andmannitol fermentation; but it was negative for the clumpingfactor reaction (CFR). It showed diffuse growth in serum-soft agar (SSA) and was phage nontypable. Electron micro-graphs showed that S-7 has a zone of an oval to roundpolymorphous vesicular structure in its outermost layer; thisis covered with a capsule layer with a zone of high electrondensity (see Fig. 3a).

Procedure for conversion experiments of freeze-dried cellsin SSA. The proportion of strain S-7 that changed from acompact to a diffuse colony morphology in SSA (conversionamount) after freeze-drying was determined by a previouslydescribed method (20). S-7 organisms were cultured in brainheart infusion (BHI) medium (Difco Laboratories, Detroit,Mich.) for 16 h (late log phase) and centrifuged at 8,000 x g.They were then harvested, and 106 cells were suspended in0.05 M phosphate buffer as controls and in two differentcryoprotectant solutions (10% nonfat skim milk [Difco] and2% honey [commercial honey from lotus-flower; Meiji Co.Ltd., Tokyo, Japan]) (11, 38) that are generally used ascryoprotectant agents by freeze-drying. The cells were thensterilized by tyndallization by the method of Ohtomo et al.(17). Then, 0.5 ml of each of these solutions was poured intoampoules (14 by 12 mm). The ampoules were frozen rapidlyin dry ice (-50°C) for 1 to 2 min and then lyophilized for 5 to48 h in a freeze-dryer (type N-77; Shimadzu Co., Ltd.,Tokyo, Japan) at 10-2 to 10' torr (mm Hg; 13.33 to 1.333Pa). After drying, 1 ml of sterile saline solution was added toeach ampoule to regenerate cultures, and 0.1 ml of theculture was mixed with 0.1 ml of normal rabbit serum, whichwas sterilized by membrane filtration (pore size, 0.45 nm;Millipore Corp., Bedford, Mass.), and 10 ml of BHI brothcontaining 0.15% agar. The cultures were then cooled at 4°Cfor 30 min and incubated at 37°C for 18 h. Cultures that were

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not freeze-dried were also prepared as controls. Colonymorphology in SSA was record.

Determination of CFR and cell volume index. The CFR andcell volume index (CVI) of the cells were determined toobserve changes in surface cell morphology caused bylyophilization by a previously described method (20). Therelative clumping factor titer was defined as the highestdilution of organisms which caused clumping, as describedpreviously (20). The CVI of the organisms was determinedas described below. Cells were harvested after 16 h ofgrowth at 37°C in plates containing BHI medium. Fivemilliliters of the cell suspension in sterile saline was placedinto Hopkins tubes and centrifuged at 3,000 x g at 4°C for 30min. Readings were taken with vernier calipers, and theheight of the column of the packed cells was recorded, inmillimeters. Also the number of CFU was estimated fromplates made from the packed cells, which were shaken withglass beads (outer diameter, 10 to 20 mm) in sterile 25-mlflasks. CVI was calculated by the following formula: [packedcell volume (milliliters)/CFU] x 101'.

Viable cell count. After the freeze-dried cells were sus-pended in sterile saline, the average viable cell count of bothsets of organisms was made in triplicate in mannitol-salt agar(Difco) by a previously described method (17).

Quantification of CSP after freeze-drying. Surface materialof strain S-7 cultures after freeze-drying was quantified by apreviously described method (20). The organisms weregrown at 37°C for 18 h in 5 liters of BHI medium and dialyzedagainst 5 volumes of distilled water overnight at 4°C. Theorganisms were then centrifuged at 10,000 x g for 15 min at4°C, 5 g (wet weight) of cells was suspended in 20 ml ofpotassium phthalate-sodium hydroxide buffer (pH 4.0) con-taining 3 g of glass beads (outer diameter, 1.00 to 1.05 mm;Glasperlen; B. Braun Melsungen Apparatebau) and blendedin a vortex mixer (Taiyo Bussan Co., Ltd., Tokyo) for 3 minat 4°C, and the cell debris was separated with a sintered glassfilter. The filtrate was centrifuged at 12,000 x g for 20 min at4°C. The supernatant was dialyzed against distilled water for48 h, lyophilized, and weighed. Also, for the quantitation ofsurface materials, values were considered to be the mean ±standard error of the means of three experiments.CAP determined by adsorption of anticapsular activities in

sera. CAP was determined by a previously described method(40). Various freeze-dried cells were cultured in BHI me-dium at 37°C for 18 h. The cells were harvested by centrif-ugation at 8,000 x g at 4°C and then lyophilized andweighed. They were combined with rabbit antiserum con-taining 1 U of activity against lyophilized cells, and theminimum number of organisms capable of complete adsorp-tion of serum activity of diffuse to compact strains in SSAwas measured. A total of 2, 4, 8, 16, 32, 64, and 128 mg oflyophilized organisms were added to 1.0 ml of anticapsularrabbit serum. These mixtures were kept at 37°C for 3 h andthen centrifuged at 7,000 x g for 15 min to remove theorganisms. The supernatant was then applied to SSA by thetechnique described above. The conversion activities of thegrowth types of strain S-7 in SSA (which contained 1 U ofanticapsular serum adsorbed with various freeze-dried cells)were determined.

Ability of the strain to adhere to fibrinogen, collagen, andlectin. Fibrinogen (human; AB; KABI, Stockholm, Sweden),collagen (type III, acid-soluble calf skin; Sigma ChemicalCo., St. Louis, Mo.), and soybean lectin (type VI; fromGlycin max; Sigma) were labeled with 125I by the chloramineT method (carrier-free-Na125I from Radiochemical Center,Amersham, Buckinghamshire, England). The specific radio-

activities of fibrinogen, lectin, and collagen were 3.70, 3.57,and 3.38 mCi/mg of protein, respectively. For the bindingassay, 4 x 109 cells of strain S-7 per ml and 1 ,ul each of'25N-labeled fibrinogen, lectin, or collagen (105 cpm) wereadded to 1.5 ml of phosphate-buffered saline, which con-tained 0.5% Tween 20 and 1% human serum albumin, andcentrifuged at 7,000 x g for 5 min. The pellets were cleanedtwice by washing each pellet in phosphate-buffered saline,and then the cell-associated radioactivity was counted in aliquid scintillation system (LS-230; Beckman Instruments,Inc., Fullerton, Calif.).Phage sensitivity. Freeze-dried strain S-7 cells were cul-

tured in BHI medium, and 30 of the colonies were selected atrandom. They were tested for phage type by using aninternational phage set by a previously described method(40).

Electron microscopy study. Strain S-7, which was freeze-dried for 24 h, was cultured in BHI medium for 18 h andcentrifuged at 7,000 x g for 5 min. The strain was fixedinitially with 7% glutaraldehyde in 0.1 M phosphate buffer(pH 1.0) at 4°C for 16 h, and then it was fixed with 0.1%osmic acid at room temperature for 5 h (20). The fixed cellswere first dehydrated with ethanol and acetone and thenembedded in Epon 812 by the usual procedure. Thin sectionswere prepared with an ultramicrotome (LKB Instruments,Inc., Rockville, Md.), mounted on carbon-coated grids,doubly stained with uranyl acetate and poststained with leadcitrate (16), and observed in an electron microscope (model10OB; Japan Optics Co. Ltd., Tokyo, Japan). S-7 cells thatdid not undergo freeze-drying were prepared in the sameway, for comparison.

RESULTSMorphological changes in SSA. All cells of the encapsu-

lated S-7 strain were of the diffuse type in SSA before theywere freeze-dried, and the viable cell count was on the orderof 106. However, 21% of the cells suspended without acryoprotectant changed from a diffuse to a compact growthtype after 10 h, and the frequency of conversion increased to75% after 24 h. In the meantime, the viable cell countdecreased. The conversion frequency in the cells that wereprocessed with 10% skim milk and 2% honey as cryoprotec-tion agents was lower than that in the control (0.05 Mphosphate buffer); it changed from 10 to 50% after 5 to 24 h(Table 1).

Effect of freeze-drying on CFR and CVI. The encapsulatedstrain of S. aureus showed no CFR in fibrinogen or plasma.However, CFR in S-7 started to increase after 10 h offreeze-drying and reached 1:512 after 24 h (Fig. 1). As CFRincreased, the ratio of conversion from a diffuse to a com-pact growth type in SSA averaged 50 to 70%, depending onthe cryoprotection agent used (Table 1), demonstrating theloss of capsules. In the meantime, CVI declined, showinggood agreement with CFR (Fig. 1).

Quantffication of CSP after freeze-drying of S-7 cells. TheCSP yield (mean + standard deviation) was 97.2 + 2.38 mg/g (wet weight) of cell before freeze-drying and changed to93.5 + 3.26, 65.3 + 2.98, and 42.9 + 1.78 mg/g (wet weight)of cell, respectively, after 5, 10, and 24 h of freeze-drying.When 2% honey was used as a cryoprotection agent, CSPwas 91.3 + 2.65, 86.7 + 2.33, and 57.8 + 1.77 mg/g (wetweight) of cell, respectively, in cells that were freeze-driedfor 5, 10, and 24 h. With 10% skim milk, the results were 89.5+ 3.09, 73.4 + 2.69, and 50.3 + 2.54 mg/g (wet weight) ofcell, respectively. Thus, in 2% honey there was a slightdecrease in CSP ability (data not shown).

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TABLE 1. Comparative effect of drying time during freeze-drying and protection agent on conversion from diffuse to compact typegrowth in SSA an encapsulated strain of S. aureus (S-7)

Effects of the following protection agentsa:Drying time Phosphate buffer (0.05 M) Skim milk (10%) Honey (2%)

D C CV (%) CFU/ml D C CV (%) CFU/ml D C CV (%) CFU/ml

0 18 ± 2e 0 ± 0 0 ± 0 18 x 106 21 ± 3 0 ± 0 0 ± 0 21 x106 32 ± 1 0 ± 0 0 ± 0 32 x 1065 14 ± 2 0 ± 0 0 ± 0 13 x 106 17 ± 2 0 ± 0 0 ± 0 16 x 106 24 ± 3 0 ± 0 0 ± 0 25 x 106

10 11 ± 1 3 ± 1 21 ± 1 14 x 105 13 ± 1 4 ± 2 24 ± 2 17 x 105 19 ± 4 2 ± 1 9 ± 1 22 x 10515 6 ± 2 7 ± 3 53 ± 3 13 x 104 7 ± 2 3 ± 1 30 ± 2 10 x 105 13 ± 2 3 ± 1 19 ± 2 16 x 10524 2 ± 1 5 ± 2 71 ± 3 7 x 103 7 ± 2 7 ± 1 50 ± 1 14 x 104 8 ± 1 7 ± 2 46 ± 1 15 x 104

a Abbreviations: D, colony counts of diffuse type growth; C, colony counts of compact type growth; CV, conversion percent; CFU/ml, viable cell counts. Theresults are the mean ± standard error of the mean of five experiments.

Effects of freeze-drying on CAP. Organisms that werefreeze-dried for different lengths of time were cultured inBHI medium, and their anticapsular sera were adsorbed toobserve the conversion from a diffuse to a compact type ofgrowth in SSA. The amount of absorption was 4.0 ± 2.0 mgbefore freeze-drying; but it increased to 8.0 ± 4.0, 16.0 +4.0, and 64 ± 8.0 mg after 5, 10, and 24 h of freeze-drying,respectively, demonstrating a correlation between theamount of adsorption and the freeze-drying period (data notshown).

Effects of freeze-drying on adhesion to fibrinogen, lectin,and collagen. The cells were cultured in different ways tocompare the adhesion of strain S-7 to different polymers.One group was freeze-dried with 0.05 M phosphate buffer(control); two other groups were freeze-dried with 10% skimmilk and with 2% honey as cryoprotection agents. In the firstgroup (0.05 M phosphate buffer), adhesion to fibrinogenincreased, but adhesion to lectin decreased. There was littlechange in adhesion to collagen in the first group after 5 to 24h of freeze-drying. The two groups that were freeze-driedwith different cryoprotectants also showed increased adhe-sion to fibrinogen, although the levels were not as high asthat in the control group. Adhesion to lectin graduallydeclined in both groups that were freeze-dried with cryopro-tectants, whereas there was almost no change in adhesion to

1:1l024i _ 30

1:51 ~~~~~~~~~~~25

E1:16-=1

* 1:1 -5O

5 10 24Freeze- drying (hr)

FIG. 1. Effect of drying time during freeze-drying on the CFR(0) and CVI (O) of an encapsulated strain of S. aureus (S-7). Eachpoint presents the mean + standard error of five experimentsperformed in duplicate.

collagen in either group that was freeze-dried with cryopro-tectant, regardless of the drying time (Fig. 2).

Effect of freeze-drying on phage sensitivity. Freeze-driedorganisms were cultured in BHI agar, and 30 of the colonieswere selected at random and tested for phage type. Beforefreeze-drying cells reacted as phage nontypable when testedwith an international phage typing set. However, after 5 h offreeze-drying, 5 of 30 colonies (16%) proved to be typable tophage 52. From 10 to 24 h, they also became typable tophage 42E. At 24 h, approximately 90% of the colonies weretypable to both phage 52 and 42E (Table 2).

_>1

,--

ct

z> 3000m 200

z 100

H

0P 400a4

300N-0" 200

Freeze-dryinig ( lir)FIG. 2. Effect of drying time during freeze-drying on adhesion to

fibrinogen (0), collagen (OI), and soybean lectin (0) of an encapsu-lated strain of S. aureus (S-7). Each point represents the mean +standard error of four experiments performed in duplicate. For thefreeze-drying methods of storage, the protection agents were 0.05 Mphosphate buffer (control) (A), 10% skim milk (B), and 2% honey(C).

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FIG. 3. Electron micrographs of an encapsulated strain of S. aureus (S-7). (a) Before freeze-drying treatment; (b) after freeze-drying for24 h. Bars, 0.1,m.

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TABLE 2. Effect of drying time during freeze-drying on phagesensitivity of an encapsulated strain of S. aureus (S-7)

Amt (%) of the followingDrying time phage typesa: Total (%)

(hr)52 42E

0 0/30 (0.0) 0/30 (0.0) 0.05 5/30 (16.0) 0/30 (0.0) 16.0

10 17/30 (56.6) 3/30 (10.0) 66.624 23/30 (76.6) 4/30 (13.3) 89.9

a Values are number of colonies tested which were sensitive to phage/totalnumber of colonies tested.

Electron microscopic observation of strain S-7 before andafter freeze-drying. When strain S-7, which was freeze-driedfor 10 h, was cultured in BHI medium and incubated at 37°Cfor 16 h, capsule or slime materials were not seen on the cellsurface (Fig. 3b). However, capsule or slime formation wasobserved in cells that were grown with the control treatment(0.05 M phophate buffer) without freeze-drying (Fig. 3a).

DISCUSSION

In many studies the mechanisms of damage to frozen orfreeze-dried cells, such as injury to cytoplasmic and outermembrane integrity (2, 6, 24, 25), disruption of activetransport and protein synthesis (8, 9, 30), damage to DNA(22, 27, 28, 37), and disruption of cell morphology (5, 10),have been reported.We examined the effects of drying time during freeze-

drying on the cell surface of an encapsulated strain of S.aureus, with particular attention paid to CSP. Cell surfacechanges were evaluated by comparison with results of pre-vious studies done with the encapsulated strain of S. aureus,S-7 (20, 39, 42). Studies of cellular characteristics such ascolony morphology in SSA, CFR, CVI, capsule antigenproduction, and phage sensitivity revealed that strain S-7turned from an encapsulated type to an unencapsulated typeafter it was freeze-dried.No investigators have pointed out that adhesion charac-

teristics are affected by changes in the outermost cell surfaceafter freeze-drying. Thus, our finding that adhesion of fibrin-ogen and soybean lectin in the freeze-dried cells was mark-edly different than that in nontreated cells indicates thatthere are additional changes in cell surface properties.Adhesion with soybean lectin, in particular, is likely to beaffected by changes in the receptor because of cellulardamage on the cell surface polysaccharide (14, 35). It isknown that the unencapsulated strain does not containN-acetylgalactosamine in the cell surface fraction (14, 19).Also, the ability of the encapsulated strain S-7 to adhere tofibrinogen increased after freeze-drying treatment. This maybe a result of exposure of surface components that binddifferently to the different cell surfaces (15).

In the process of freeze-drying, drying is known to ad-versely affect organisms; viable cell count declines markedlywhen intracellularly bound water is dehydrated (6, 8, 23),which is probably caused by damage to the permeability ofthe cytoplasmic membrane (30) and denaturation or confor-mational change of enzyme and protein (6). Results of thisstudy demonstrated that as the lyophilization time increased,the number of viable cells declined, and at the same time,more changes were observed on the cell surface. These datasuggest that the rehydration of bacterial cells can introducevarious types of variation, such as metabolic injury or

genetic damage (27). In our previous studies (17) of changesof colony morphology in SSA, we showed that drying affectscells more than freezing does. We suspect that dryingsubstantially affects the outermost cell surface, but it isnecessary to examine precisely how drying affects the cellsurface, especially capsule or slime, compared with theeffects of freezing. Further studies are needed, however, todetermine whether dehydration affects capsule or slimebiosynthesis on the plasmid and chromosome at the geneticcontrol level (16, 31) or whether it injures the cell membraneand cell wall (23, 30).We have already reported that CSP is inhibited by high-

temperature cultivation (13) or the addition of bile acidderivative (12, 20). Loss of CSP because of freeze-drying isan important issue because it affects the maintenance ofmorphological stability during the preservation of microor-ganisms (1, 8, 26, 29). The biological significance of capsuleor slime has attracted attention in various fields recently (3,4, 7). Freezing with a cryoprotection agent and drying havetraditionally meant that the cell walls and membrane areprotected (8, 33). We found that use of different cryoprotec-tion agents (10% skim milk and 2% honey) results in differentlevels of cell surface stability, which indicates that selectionof a proper cryoprotection agent determines how well thecell surface can be preserved. Additionally, for the success-ful preservation of encapsulated bacterial strains, a numberof problems remain to be solved to maintain stability andhigh product yield, for example, for the preservation of stockcultures for vaccine production (5).The encapsulated strain of S. aureus is known to be phage

nontypable (39). Nevertheless, Sompolinski et al. (32) havereported that the encapsulated strains of S. aureus becomephage typable in the presence of calcium chloride. In ourstudy, the phage-nontypable strain S-7 became typable withthe prolongation of the lyophilization time; after 5 h, somecells of S-7 were typable to phage 52, and after 10 h manycells were also typable to phage 42E. This demonstrates thatthere is restricted capsule production on the surface of cells,as well as changes in the phage receptor, after prolongedperiods of lyophilization.

In conclusion, results of this study indicate that freeze-drying affects the cell surface of the encapsulated strain of S.aureus, particularly CSP. This finding will be valuable forstudying the mechanisms of capsule or slime biosynthesisand the conditions necessary for maintaining a stable outer-most cell surface. The mechanism of how CSP is inhibited byfreeze-drying in S. aureus is currently under investigation inour laboratory.

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