Avirulence and Immunogenicity in Mice of a Bovine MastitisStaphylococcus aureus Mutant

Cristina Bogni, Mariela Segura, Jose Giraudo, Ana Giraudo, Aldo Calzolari, and Rosa Nagel

ABSTRACT

An avirulent mutant, designatedRC122, was derived from Staphylo-coccus aureus bovine mastitis strainRC108 after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis.Mutant RC122, which was isolatedon the basis of reduced colony size,showed diminished virulence inmice (LD50 of RC122: 3.1 X 1010 cfuvs LD50 of RC108: 2.3 X 107 cfu).Mutant RC122 grew more slowlythan its parental strain and showeddecreased production of severalexoproteins, such as a- and B-hemolysin, DNAse and coagulase.The production of its capsule wasinduced only under in vivo growthconditions. Clearance studies per-formed in the mouse kidneyrevealed that the kinetics of disap-pearance of the mutant was similarto that of its parental strain. Protec-tion experiments carried out byintraperitoneal administration inmice showed that mutant RC122conferred a good degree of protec-tion from challenge with homolo-gous and heterologous strains.

RESUME

Un mutant avirulent (RC122) deStaphylococcus aureus fut obtenu apartir de la souche de mammitebovine RC108 par mutageneseavec le N-methyl-N'-nitro-N-nitrosoguanidine. Le mutant RC122etait moins virulent pour la sourisque la souche RC108 (DL50 de 3,1 X1010 et 2,3 x 107, respectivement).Le mutant RC122 avait une crois-

sance plus lente que la soucheparentale, de meme qu'une produc-tion plus faible de plusieurs exopro-teines telles que les hemolysines ax etB, la DNase et la coagulase. La pro-duction de capsule d'etait induiteque dans des conditions de crois-sance in vivo. Des etudes d'elimina-tion effectuees au niveau de rein desouris ont demontre que la cine-tique d'elimination du mutant etaitsimilaire a celle de la soucheparentale. Des etudes de protectionpar injection intraperitoneale dumutant RC122 ont demontre quecette souche conferait un bonniveau de protection contre dessouches homologue et heterologues.

(Traduitpar docteur Serge Messier)

INTRODUCTION

Staphylococcus aureus, a bacterialpathogen that produces localized anddisseminated infections in man andanimals, is the main etiological agentof bovine mastitis. This diseasecauses serious economical lossesworldwide (1).Mutants of S. aureus affecting the

synthesis of pathogenicity determi-nants have been isolated after chemi-cal, physical or transposon inducedmutagenesis (2-12). Most of themutants of this type, even those due toa single mutation, are pleiotropic,affecting to different degrees the syn-thesis of several exoproteins. Thesemutants showed different degrees ofavirulence (6,7,10-12).Up to now, several vaccines against

bovine mastitis have been developedon the basis of cell extracts, capsular

polysaccharides and/or exotoxins(13-15). One such a vaccine, based oncell extracts and capsular polysaccha-rides, has been recently developed inour laboratory and found to be effec-tive in significantly reducing the rateof clinical and subclinical bovinemastitis (16,17). However, on theoret-ical grounds it could be assumed thata vaccine based on a live, avirulentstaphylococcal strain could be moreeffective (through limited multiplica-tion) in conferring a stronger andlonger lasting immunity against infec-tion. With this purpose in mind, asearch for avirulent S. aureus mutantswas undertaken.

In this work we describe the isola-tion and characterization of an aviru-lent mutant isolated from a S. aureusbovine mastitis strain that confersimmunity in mice against homologousand heterologous virulent S. aureusstrains.

MATERIALS AND METHODS

Bacterial strains: S. aureus RC18,isolated from a subclinical bovinemastitis case from a dairy cow inCordoba, Argentina (18); S. aureusRC108, a spontaneous streptomycinresistant (Smr) mutant derived fromstrain RC18, and synthesizes aninducible capsule of antigenic type A(19); S. aureus strain Smith, constitu-tive for the production of a capsule ofantigenic type A (20), sent byDr. Hash (Vanderbilt UniversitySchool of Medicine, Nashville,Tennessee, USA); S. aureus RC46,isolated from a clinical bovine masti-tis case in a dairy in Cordoba (8).

Departamento de Microbiologia e Inmunologia, Facultad de Ciencias Exactas, Fisico, Quimicas y Naturales (Bogni, Segura, Giraudo A., Calzolari,Nagel), Departamento de Patologia Animal, Facultad de Agronomia y Veterinaria (Giraudo J.), Universidad Nacional de Rio Cuarto (5800) RioCuarto, C6rdoba, Argentina; CEFYBO-CONICET, Serrano 665 (1414) Capital Federal, Argentina (Nagel).Corresponding author: Dr. R. Nagel, Departamento de Microbiologia e Inmunologia, Facultad de Ciencias Exactas, Fisico, Quimicas y Naturales,Universidad Nacional de Rio Cuarto (5800) Rio Cuarto, C6rdoba, Argentina.Received Octobre 7, 1997.

Can J Vet Res 1998; 62: 293-298 293

MUTAGENESIS

Broth cultures of S. aureus RC108were grown to logarithmic phase in5 mL of brain-heart infusion broth(BHI, Difco), centrifuged and resus-pended in 4 mL of citrate buffer.N-methyl-N'-nitro-N-nitrosoguanidine(MNNG) was added to the bacterialsuspension to a final concentration of100 jig/mL. The mixture was incu-bated in a 37°C water bath for 30 min.After MNNG treatment, aliquots of100 ,uL, each containing about104 cfu/mL surviving cells, were inoc-ulated into 700 uLL of BHI broth andincubated for 4 h at 37°C. Dilutions ofthese cultures were spread on BHIagar plates and incubated for 18 h at39°C. Small colonies were isolatedand kept at -200C in 20% glycerol.Individual colonies were grown inBHI broth for 72 h at 39°C. Balb-cmice (produced in the UniversidadNacional de Rio Cuarto from stocksprovided by the Centro Panamericanode Zoonosis, Argentina), 21 to 25 dold, were inoculated by the intraperi-toneal (IP) route, with 1 mL of eachof these cultures (approximately109 cfu/mL). The mice were observedduring 72 h and the results wererecorded as death or not death.

REVERSION EXPERIMENTS

Two procedures were followed fordetection of revertants: (1) logarith-mic cultures were centrifuged andresuspended in 4 mL of citrate buffer.Ethyl methanesulfonate (EMS) wasadded to the bacterial suspension to afinal concentration of 100 ,ug/mL.The mixture was incubated in a 37°Cwater bath for 30 min. Aliquots of100 p,L containing about 104 cfu/ sur-viving cells were inoculated in700 ,L1 of BHI broth and incubatedfor 4 h at 37°C. Dilutions of these cul-tures were spread on BHI agar platesand incubated for 18 h at 370C.One mL of 72-hour cultures of each of250 colonies (with a size similar tothat of the parental strain) wereinjected IP into mice (2). Aliquots of1 mL of a 72-hour culture, obtainedafter 20 succesive serial subculturesof RC122 in BHI broth, were injectedto each of 4 mice.

DETERMINATION OF THE LD50

Cells were grown in BHI broth over72 h at 37°C, centrifuged (4000 x gfor 10 min) and resuspended in BHI

broth. Balb-c mice, 21 to 25 d old,were inoculated IP with 1 mL of 6 dif-ferent concentrations of the strain (104to 1010 cfu). When necessary, the 72-hour cultures of the mutant were con-centrated (up to lOX or IOOX).Groups of 5 mice were used for eachconcentration of bacteria. Theseexperiments were done in duplicate.Data were recorded after 72 h. TheLD50 was calculated by the Reed andMuench method, as described (10).

MICROBIOLOGICAL TESTS

Quantitative determination of a-and B-hemolysin, coagulase andDNAse was carried out as describedpreviously (8,9,11). Briefly: cellsgrown in BHI broth on a rotatoryshaker at 37°C for 15 h were cen-trifuged for 15 min at 11 000 X g at4°C and the supernatants were dilutedusing 1% washed rabbit and sheeperythrocytes, rabbit plasma, and tolui-dine-DNA-agar, respectively.

For 8-hemolysin identification twomethods were used: (i) hemolysis onhorse blood agar and (ii) hemolysis onsheep blood agar. For this latter test aculture of Corynebacterium ovis wasstreaked on a blood agar plate, incu-bated for 24 h at 37°C, cross-streakedwith cultures of the strains to beassayed, and further incubated at37°C. Corynebacterium ovis producesdiffusible substances that inhibit theactivities of ot- and B-hemolysins (21).Lipase and protease production,

mannitol utilization, phosphataseactivity and presence of clumping fac-tor were assayed, as described previ-ously (10,18).

In vitro induction of the productionof the capsule was assessed by the dif-fuse colonial morphology in serum-soft agar (ml l0-SSA), pH 6.0, afterthree successive passages throughBHI broth supplemented with 10%bovine serum (22,23). The productionof a capsule in vivo was assessed inmlIO-SSA, pH 7.0, with bacteriarecovered in 9 mM NaCl from theperitoneum of mice inoculated 8 hbefore the sampling (23).Growth curves were determined

with cultures grown in BHI broth orchemically defined medium (CDM)(24).

PLASMID AND PROTEIN ANALYSES

Plasmid DNA was extracted by amodified alkaline method (25). Extra-

and intracellular protein profiles weredetermined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis(SDS-PAGE) as described (8,9).

ORGAN INVASIVENESS IN MICE

The procedure of Lee et al (4), wasfollowed. Briefly: S. aureus strainsRC108 and RC122 were grown for18 h on BHI broth and their OD640values were adjusted so that theappropriate bacterial concentrationswere obtained. Mice were injected IPwith -2.0 X 107 cfu of eitherS. aureus RC108 or RC122. At vari-ous intervals after injection, groups of8 to 15 mice were sacrificed by etherinhalation and the kidneys wereexcised, weighed and emulsified in1 mL of saline solution, using ahomogenizer. Ten-fold dilutions ofthe tissue homogenate were done andspread on BHI agar plates in dupli-cate. The number of cfu/mL of thehomogenate was used for calculatingthe total number of cfu/g of tissue. Insome experiments, samples fromblood, spleen, lungs and liver werealso processed and plated on BHI agarplates.The ANOVA was applied to com-

pare means of data.

PROTECTION EXPERIMENTS

Groups of 25 mice (weighing 13 to15 g) were inoculated twice, IP, witha 15-day interval between inocula-tions. For homologous protection,each group received graded doses(from 106 to 109 cfu) of a culture ofmutant RC122 suspended in peptonewater (0.5% w/v peptone, 0.5%NaCl). Seven days after the 2nd dose,mice were challenged by IP injectionwith 5 different doses, from 105 to108 cfu of the RC108 strain. For het-erologous protection, groups of25 mice were inoculated as beforewith the dose of 108 cfu and chal-lenged with RC46 or Smith strains.Groups of mice inoculated with pep-tone water were used as control. TheLD50 was determined as indicated.

RESULTS

ISOLATION AND SELECTION OFAVIRULENT MUTANTS

One hundred thirty independentclones showing a reduced size after18 h growth on BHI agar plates at

294

TABLE I. Phenotypic characteristics of mutant RC122 and parental strain RC108

Characteristica-hemolysin (U/108 cfu)P-hemolysin (U/108 cfu)8-hemolysin (U/108 cfu)Coagulase (U/10b cfu)DNAse (U/1I' cfu)LipaseCatalasePhosphataseProteaseMannitol utilizationPigment productionClumping FactorColonial morphology in ml 10-SSAat pH 7.0at pH 6.0at pH 7.0 after in vivo growthResistance to streptomycinResistance to chloramphenicol

RC1083.8120+

488492 000

+

+++

C

DDRR**

RC1221.332

37177 000

+*+

+

C

C

DRR

D = Diffuse; C = Compact (colonial morphology in ml 10-SSA medium); R = resistant to anti-biotics. (*) Halos slightly smaller than those of strain RC108. (**) Conferred by a 2.8-kb plasmid

TABLE II. Generation time of strain RC108 and mutant RC122 after growth under differentconditions

Conditiona RC 108 RC122BHI, 37°C, with agitation 19 ± 0.5b 23 ± 0.4BHI, 37°C, without agitation 33 ± 0.5 45 ± 1.2BHI, 43°C, without agitation 36 ± 1.5 46 ± 3.0CDM, 37°C, without agitation 103 ± 2.2 122 ± 3.1a BHI = Brain Heart Infusion broth; CDM = Chemical Defined MediumbData are mean of at least 2 experiments

Figure 1. SDS-PAGE of exoproteins fromparental strain RC108 and mutant strainRC122 after growth without or with agita-tion. M = Molecular weight markers (kDa).

39°C were isolated after treatment ofstrain RC108 with MNNG. Theseclones were assayed for virulence byadministration of 1 mL of 72-hourcultures in BHI broth with a concen-

tration 30- to 40-fold higher than thatcorresponding to the LD50 of theparental strain RC108. Five out of130 clones did not kill the treatedmice.The LD50 determination of these

5 clones showed that they were 40 to1350 times less virulent than strainRC108. Clone RC122, which showedthe highest decrease in virulence(LD50 of 3.1 X 1010 cfu, as comparedto the LD50 of RC 108 of 2.3 X107 cfu), was chosen for further study.

PHENOTYPIC CHARACTERIZATION

The production of several exopro-teins and other phenotypic traits ofmutant RC122 and of its parentalstrain are seen in Table I. The mutantshowed a reduced production of mostof the exoproteins analyzed; coagu-lase production was the one the mostdrastically reduced. No differenceswere detected between the mutantRC122 and parental strain RC108 inthe synthesis of catalase, phosphatase,

production of pigment and presenceof clumping factor. Mutant RC122showed a slower rate of oxidation andfermentation of mannitol.The SDS-PAGE protein profile of

the supernatants of both mutant andparental strains were compared. Asshown in Fig. 1, many proteins fromthe supernatant of cultures of strainRC122 grown in BHI broth withoutagitation were diminished or almostmissing. In contrast, the protein pro-

files from supernatants of culturesgrown in BHI broth with agitationwere similar for both strains.

In contrast with its parental strain,mutant RC122 did not show diffusecolonial morphology in ml 10-SSA,pH 6.0. However, cells of RC122recovered from the mouse peritoneumdid induce the diffuse colonialmorphology in mllO-SSA, pH 7.0(Table I).Mutant RC122 displayed the same

plasmid profile as its parental strain.Three bands of approximately 45, 30and 2.8 kb were observed with bothstrains (Fig. 2).The rates of growth of both strains,

parental and mutant, were comparedin BHI and CDM media under differ-

Figure 2. Agarose gel electrophoresis of DNAof parental strain RC108 and mutant strainRC122. 0 = Origin. C = Chromosomal DNA.p = plasmids bands. The numbers indicatethe approximate molecular weight (kb).

ent conditions. As seen in Table II,mutant RC122 showed an increasedgeneration time when grown in eithermedium at 37°C or 43°C.To assay for reversion, 250 colonies

from an EMS-mutagenized culture ofmutant RC122, having a colony size

295

.__r

-:- .o..; _,

*-. P-

6-

5-

SOc

I.-0

0)

I-

0)0.1

4 -

3-

2-

1 -

0 -

T 1T *

T

1

RCI108- RC122

3 6 9 12

DaysFigure 3. Recovery of viable S. aureus from kidneys of mice after intraperitoneal inoculationwith parental strain RC108 or mutant strain RC122. Each data were the average of 7 to15 kidneys. Each kidney was excised from a different mouse. Data are the mean + SD log cfu/gof kidney. (*) P = 0.014.

similar to RC108, were picked andgrown in BHI broth. None of thesecultures were found to be virulentwhen tested in mice. Likewise,aliquots of a 72-hour grown culture ofstrain RC122 that had been seriallytransferred through 20 passages inBHI broth, did not had any lethaleffect when assayed by intraperi-toneal route in mice.

ORGAN INVASIVENESS

A mouse infection model was usedto determine the invasiveness ofmutant RC122. As shown in Fig. 3,the parental strain could be isolatedfrom kidneys up to 9 d after IP admin-istration. The mean viable countsdecreased from 5.7 to 1.4 log cfu/g ofkidney. Mutant strain RC122 couldalso be cultured from kidneys 9 dafter infection. In all the samples, themean viable counts of strain RC122were lower than those of RC108,though this difference was not signifi-cant, except for the 6-day sample (Fig. 3).

The ability of both strains, parentaland mutant, to disseminate to otherorgans (spleen, lung, liver and blood)was also evaluated. Both strains wererecovered from all the organs up to4 d after administration (data notshown).

PROTECTION EXPERIMENTS IN MICE

The ability of mutant RC122 toconfer protection to mice from chal-lenge with the wild type strain RC108was analyzed as described in Materi-als and Methods. During the immu-nization period, the immunized mice(not even those inoculated with109 cfu), did not form abscesses at theinoculation site or show any othersign of disease. Seven days after the2nd injection, mice were challengedintraperitoneally with different dosesof parental strain RC108. Table IIIshows the ratios of the LD50 of immu-nized vs untreated mice. The maximalratio was obtained when the micewere immunized with 109 cfu. The

TABLE III. Protection experiments withmice immunized with mutant RC122 andchallenged with the homologous strainRC108

Immunization LD50 (cfu) ofdose of mice challenged ApproximateRC122 with RC108 Ratioa

2.3X 107106 2.2 x 107 1107 2.2 x 108 10108 6.6 x 108 30109 8.9 x 108 40

Data are the average of at least two experimentsa LD50 of immunized mice versus LD50 of unim-munized mice

TABLE IV. Protection experiments withmice immunized with mutant RC122 andchallenged with heterologous strains

LD50 ofunimmunized

LD50 ofimmunized

Strain mice (cfu) mice (cfu) RatioaRC46 7.9 X 107 5.9 X 108 7.5Smith 3.1 x 106 2.8 X 108 90Data are the average of at least two experi-ments. Mice were immunized with two doses of108 cfu of mutant RC 122a LD50 of immunized mice vs LD50 of unimmu-nized mice

minimal dose that conferred protec-tion to mice from challenge withRC108 was 107 cfu.The protection conferred by the

mutant to the challenge with two het-erologous strains was also investi-gated. As shown in Table IV, theLD50s of strains RC46 and Smith withmice that were immunized with themutant were found to be 7.5 and90 times higher, respectively, than theLD50 of the untreated mice.

DISCUSSION

Mutant RC122, described in thiswork, was isolated on the basis of asomewhat reduced colony size at 39°Cand avirulence in mice. The rationaleof this approach was based on theassumption that diminished growthrate might also affect virulence (10).In fact, that 4% of the colonies withreduced size tested turned out to beless virulent. One of these mutantclones, designated RC122, that wasabout 1350 times less virulent than itsparental strain, was further character-ized. Mutant RC122 showed a longergeneration time in BHI broth at 37°Cand 43°C (Table II) and producedslightly diminished levels of ox- and B-hemolysins and DNAse and

296

pronounced diminished levels ofcoagulase (Table I).SDS-PAGE of exoprotein profiles

from the supernatant of cultures of themutant grown in BHI broth withoutagitation revealed that many bandsgave a lower intensity or were almostmissing (Fig. 1). This observationindicates that mutant RC122 differsfrom its parental strain by the loweredproduction of many exoproteins.The loss of virulence of mutant

RC122 could not be ascribed to a lossof plasmids, since it harbours thesame three plasmids carried by itsparental strain (Fig. 2). Plasmids ascarriers of virulence determinants instaphylococci appear to be limited upto now to those coding for determi-nants of the exfoliative toxin (26),enterotoxin D (27) or an adhesion fac-tor (28).

Several characteristics of S. aureuswere found to be associated with viru-lence, such as production of hemo-lysins, coagulase, DNAse, presence ofa capsule, etc. (2,4,5,10,29). How-ever, a strict correlation between anyof these characteristics and virulencehas not been definitively established.Thus, the coagulase-negative mutantsdescribed by Hasegawa and SanClemente (2) were about 50 times lessvirulent by IP assays in mice. Othermutants defective in the production ofa single exoprotein character, such asprotein A or ct-hemolysin, or affectedin genes controlling the synthesis ofseveral exoproteins, such as agr, saror sae, were found to be 2 to 10 timesless virulent than the correspondentparental strains (3,5-7,11). Moreover,some mutants have been describedwhich, in spite of showing reduced ornull production of coagulase and/orother exoproteins, retained their viru-lence in mice (10,30). Hence, dimin-ished production of these exoproteinsdoes not appear to be the sole cause ofthe drastically diminished virulenceof mutant RC 122.

Interestingly enough, a correlationbetween longer generation time andlowered virulence, independently ofthe capacity to synthesize several exo-proteins, was observed by Odiernoet al (10) with some S. aureusmutants. However, it must be notedthat the generation time of RC 122 wasmuch shorter than that of very slowgrowing, but less attenuated, mutants(10). Notwithstanding, the lower

growth rate of mutant RC 122 mightbe an additional cause of its lowervirulence.Adherence of bacteria to host tis-

sue, through interaction with fibro-nectin, laminin, fibrinogen, etc isanother important step in the develop-ment of infection favoring coloniza-tion and tissue invasion (31,32). Sincecells of the mutant RC 122, like thoseof its parental strain RC108, wererecovered from all the organs and flu-ids analyzed, one can infer that thismutant retains the capacity to attachto host cell tissues, to invade organs,and probably to multiply in them.Clearance studies carried out in thekidney revealed that the kinetics ofdisappearance of the mutant was simi-lar to that of its parental strain(Fig. 3).No virulent revertants of RC122

were found after successive culturingof the mutant. Attempts to obtain vir-ulent revertants after EMS mutagene-sis were also unsuccessful, since nosuch revertants were isolated among250 clones screened showing a colo-nial size similar to that of the parentalstrain.At present, the avirulence of mutant

RC122 cannot be ascribed to one sin-gle defect. One could speculate thatits avirulence could also be due to ofan as yet unidentified virulencefactors.The degree of protection conferred

by intraperitoneal administration ofmutant RC122 to a challenge with thewild type strain RC108 was also stud-ied. The LD50 of RC108 in miceimmunized with different doses of themutant was about 10 to 40 timeshigher than that of untreated mice(Table III). Moreover, mutant RC122not only conferred protection to micefrom the challenge with the parentalhomologous strain RC108, but alsofrom the challenge with two heterolo-gous strains tested, a wild type strainisolated from a clinical mastitis caseand the highly encapsulated Smithstrain (Table IV). The immunity con-ferred to mice by mutant RC122 fromthe challenge with the highly encap-sulated strain Smith might result fromthe fact that this mutant is able toinduce in vivo the production of thecapsule, which like that of Smithstrain, is of antigenic type A (19).Immunization to homologous

strains by treatment with live-attenu-

ated mutants of S. aureus has beenreported previously (2,10,14). It hasalso been reported that followingimmunization with a live S. aureusattenuated vaccine, sheep develop astronger anti-S. aureus antibodyresponse than those non-immunizedor immunized with a conventionallyprepared killed S. aureus vaccine(33).

Preliminary experiments indicatethat inoculation in the bovine mam-mary gland with a high titre of cells ofmutant RC122 does not inducemastitis.

Altogether, the fact that mutantRC 122 is markedly avirulent and thatit confers in mice immunity againsthomologous and heterologous highlyvirulent strains, makes it an interest-ing candidate for the preparation andassay of a live-attenuated vaccineagainst staphylococcal infections.Experiments aimed at determining theimmunization properties of mutantRC122 against bovine mastitis areunderway.

ACKNOWLEDGMENTS

We thank DVM Alberto Ramponeand E. Reinoso for collaborating insome of the experiments. We aregrateful to M. Ballesteros and S.Saldanlo for technical assistance.

This work was supported by grantsfrom the Consejo Nacional de Investi-gaciones Cientificas y Tecnicas(CONICET), and Secretaria deCiencia y Tecnica de la UniversidadNacional de Rio Cuarto. R. N. is aCareer Researcher of CONICET. M.S.is a fellowship from UniversidadNacional de Rio Cuarto.

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