JOURNAL OF BACTERIOLOGY, Mar. 1969, p. 1343-1351Copyright © 1969 American Society for Microbiology

Salmonella abony-Salmonella typhimuriumRecombinant Nonvirulent for the Mouse

V. KRISHNAPILLAI1 AND K. KARTHIGASU2

Department of Bacteriology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia

Received for publication 21 November 1968

A previous genetic investigation involving a mouse-nonvirulent Salmonella abonydonor (high frequency of recombination) and a virulent S. typhimurium recipientindicated that two unlinked "low-virulence" loci determined nonvirulence. Anonvirulent recombinant was analyzed to determine the basis for its nonvirulence.The recombinant was smooth (like the parental strains) and prototrophic. Thedoubling time in mouse serum of the recombinant and the S. abony parent (bothstreptomycin-resistant) was longer than that of the wild-type streptomycin-sensitiveancestor of the S. typhimurium recipient. The virulent recipient also grew poorly inserum. However, the nonvirulence of the recombinant was probably not due to its in-heritance of the streptomycin-resistance allele from the donor, because other re-combinants were streptomycin-resistant but still virulent. Unlike the nonvirulentS. abony (but like the S. typhimurium), the recombinant was insusceptible to rapidintravenous clearance in normal mice. It therefore appears that neither of the"low-virulence" loci determine iminished virulence by enhancing phagocytosis.Clearance of the recombinant was enhanced by opsonization with immune serum.Counts of viable bacteria in the blood, liver, and spleen of normal mice after intra-venous challenge showed that the recombinant, like the S. abony donor, failed toproliferate in the tissues, whereas the virulent S. typhimurium did so markedly. It isconcluded that the nonproliferation of the recombinant was determined by one orboth of the "low-virulence" loci from the nonvirulent S. abony donor.

There are two regions of the bacterial chromo-some which specify nonvirulence for mice (i.e.,inability to kill with a few bacteria) of a smoothstrain of Salmonella abony (11). This was inferredfrom the segregation of nonvirulence in crossesbetween the nonvirulent strain of S. abony(LD6o = 108 bacteria intraperitoneally) as thechromosomal Hfr (high frequency of recombina-tion) donor and a virulent strain of S. typhi-murium (LD5o = <50 bacteria) as the recipient(F-). One nonvirulence locus was mapped be-tween the gene for inositol fermentation (inl)and the gene controlling one of the steps in thebiosynthesis of methionine (metA), and the otherwas mapped between the gene controlling high-level resistance to streptomycin (strA) and one ofthe genes (aroD) specifying the biosynthesis ofaromatic amino acids (11, 14, 17). Recombinantswhich inherited either of these two loci were ofintermediate virulence, i.e., with LDIo values

I Preent address: Faculty of Science, Monash University,Clayton, Victoria, Australia 3168.

2 Present address: Department of Microbiology, School ofMedicine, University of Western Australia, Perth, Westem Aus-tralia 6000.

between those of the S. abony and S. typhimuriumstrains. When a recombinant of intermediatevirulence was backcrossed to the S. abony donor,an F2 recombinant, which also inherited thesecond nonvirulence locus of the donor, becameas nonvirulent as the S. abony parent.

This paper compares various characteristicsof a completely nonvirulent Salmonella recom-binant with those of its parental strains, in anattempt to understand the physiological basis ofits nonvirulence.

MATERIALS AND METHODSCulture media. Nutrient broth consisted of Lab-

lemco Beef Extract (Oxoid), 0.5%; Bacto-Peptone(Difco), 1%; and NaCl, 0.5%. Nutrient agar wasnutrient broth solidified with 2% agar (Difco).

Diluent. Phosphate buffer (0.1 M, pH 7.4) was used.Bacterial strains. Table 1 shows the characteristics

ofthe strains used. All the strains have the Kauffmann-White 0 antigens 1, 4, 5, and 12. S. abony SW1444is an Hfr strain and probably identical to S. abonySW1391 (13). S. typhimurium CS is the mouse-virulentprototrophic wild-type strain described previously (6).S. typhimurium M206 is mouse-nonvirulent and sensi-tive to the bactericidal action of serum (12). S.

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1344KRISHNAPILLAI AND KARTHIGASU

TABLE 1. Characteristics of strains

Genotype0 MouseStrain vn(o,bac- Source or origin Refer-

his metA aroD i#l rha ara strA Hi H2 teria/mouse)

SW1444 + _ - + + + r b e, n, x 5 X107 P. H. Makeli 11, 14C5 + + + + s i 1, 2 <10 D. Rowley 6CSP - + + s i 1, 2 <10 C5 11M206 + + + - + s i 1, 2 5 X 10" D. Rowley 6C5R + + + + + - r b 1, 2 107 SW1444 X CP 11Lister + + + + + + s b 1, 2 106 R. Bhagwan Singh -

Abbreviations (17): his, metA, and aroD denote genes determining biosynthesis of histidine, methio-nine, and aromatic amino acids (tyrosine and phenylalanine), respectively; inl, rha, and ara denotegenes specifying utilization of inositol, rhamnose, and arabinose, respectively; strA denotes gene con-trolling streptomycin resistance (1 mg/ml); HI and H2 denote genes specifying synthesis of flagellarantigens of phase 1 and phase 2, respectively; + = ability to synthesize or utilize; -= inability tosynthesize or utilize; r = resistance; s = sensitivity.

typhimurium C5P is a double mutant selected from C5and previously described as C5 his ara (11). C5R isthe completely nonvirulent recombinant obtained byrecombination between SW1444 as donor and C5Pas recipient (11). It was derived as follows. A recombi-nant selected for the donor his+ allele also inheritedthe donor strA resistance allele. Its virulence was inter-mediate between that of the donor and the recipient(LDIo = 105 bacteria), as previously reported (11).This recombinant was then crossed with the S. abonydonor, selection being made for the rha+ allele of thedonor. C5R, the rha+ recombinant from this back-cross, also inherited the inl+ and HI-b loci from thedonor as unselected markers. Its mouse virulence wassimilar to that of the donor (LD6o = 108 bacteria, i.e.,nonvirulent), as previously reported (11). The re-cipient alleles still present in the recombinant wereara and H2-1, 2. This strain will be referred to as re-combinant C5R. S. paratyphi B Lister was the strainused in the Institute for Medical Research, KualaLumpur, for the preparation of typhoid-paratyphoidvaccine.

Preparation of bacterial cultures. Nutrient brothcultures, incubated for 18 hr at 37 C without shaking,were used in all experiments.

Mice. Random-bred Swiss white mice (of bothsexes) weighing 18 to 22 g, obtained from the Institutefor Medical Research, Kuala Lumpur, were used.

Mouse virulence of bacterial strains. Broth cultureswere diluted and 0.2-ml dilutions were injected intra-peritoneally into groups of 10 mice. The mice werehoused in groups of 10. Mortality was recorded dailyfor 28 days, and the LD5o dose was calculated (15).

Clearance of viable bacteria from the mouse blood-stream. The technique was similar to that describedpreviously (1, 9), except that recovery of viable bac-teria was measured rather than recovery of uP label.Heparin was not used, because it has been reportedthat there is no significant difference in the rate ofclearance of bacteria in normal and heparinized mice(1). A sample (0.2 ml) of a broth culture was injectedinto a tail vein (5 X 107 to 10 X 107 bacteria/mouse).At intervals, 0.05 ml of blood was obtained from the

retro-orbital venous plexus with a calibrated glasspipette, diluted, and plated in duplicate on nutrientagar. For each bacterial strain, the logarithm of thearithmetic mean of the counts from three to five micefor a particular time was plotted against time, and thebest-fit straight line through the points was calcu-lated; its slope, which measures the rate of clearance,is the "phagocytic index" (K) and may alternatively becalculated from the equationK = (log,oC, -log1oC2)/(T2- T1), where Cl and C2 are the counts at times T1and T2 in minutes (9). The calculations were made byusing an IBM360 model 50 computer at StanfordUniversity School of Medicine. The K values are ex-pressed with two standard errors of the slope. In con-trol experiments, it was determined that the percentagerecovery of viable bacteria immediately after inocula-tion (assuming a mouse blood volume of 2 ml) was75 to 95% of the inoculum.Mouse serum. Normal mice were bled from the

retro-orbital venous plexus and the blood was pooled.After clotting, the serum was collected by centrifuga-tion and stored at -20 C. To obtain immune mouseserum, animals were inoculated intraperitoneally with106 viable bacteria of the immunizing strain, and werebled 2 weeks later.

Opsonization of bacteria. One milliliter of undilutedimmune serum and an equal volume of broth culturewere mixed and held at 4 C; after 20 min, 0.4 ml of thismixture was inoculated intravenously into mice andthe rate of clearance was determined as described.Immune mice. Mice were inoculated intraperitone-

ally with 101 viable bacteria of the nonvirulent strainC5R. Twelve days later they were used in clearancetests.

Liver and spleen counts. Groups of mice were inocu-lated intravenously in the tail vein with 5 X 106 to10 X 106 bacteria/mouse. At intervals, three mice foreach bacterial strain were killed and their livers andspleens were removed aseptically. They were homoge-nized at 2,000 rev/min for 60 to 90 sec in a Stir-Rtissue homogenizer (Tri-R Instruments, Jamaica,N.Y.). Samples of diluted homogenate were spread onagar in duplicate for viable count.

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Bacterial growth rate in normal mouse serum. Brothcultures diluted in buffer were mixed with undilutedserum to obtain two concentrations of serum: 90%(9 parts serum, 1 part bacterial suspension) and 10%(1 part serum, 9 parts bacterial suspension). Theamount of broth carried over during dilution was0.1% into the 90% serum-mixture and 0.9% into the10% mixture. The initial bacterial concentrations were

adjusted to about 105 bacteria/ml. The mixtures were

held in a water bath at 37 C, and at intervals sampleswere diluted and plated on agar in duplicate for viablecount.

RESULTS

Smooth-rough character. The smooth or roughcharacter of Salmonella strains can be determinedby the pattern of resistance to phages which dis-tinguish the qualitative nature of the bacteriallipopolysaccharide (18). The strains shown inTable 1 were tested with the phages used in B. A.D. Stocker's laboratory. The parental strainsC5P and SW1444, and the recombinant C5R,were fully smooth (Table 2), as were C5 (wild-type ancestor of C5P) and S. paratyphi B Lister.The insensitivity of strain Lister to phages P22and P22h was presumed to be due to its lysogeny.Strain M206 was sensitive to several rough-specific phages, which suggests that this strainwas at least partly rough.Growth rate in normal mouse serum. Certain

streptomycin-resistant mutants of a mouse-virulent strain of S. typhimurium have a 3 to 7min longer doubling time in nutrient broth, andare less virulent than the parent (7). One suchmutant was found to be less capable of pro-liferation in the mouse than was the virulentparent (7). We therefore tested the recombinantand the parental Salmonella to determine whetherthey differed in growth rate, since the virulentparent C5P was streptomycin-sensitive, whereasthe other parent, SW1444, and the recombinant,

C5R, were both nonvirulent and streptomycin-resistant. Growth in mouse serum would be acloser analogy to growth in the host than growthin nutrient broth, and so normal mouse serumwas used at two concentrations (90 and 10%),with the 90% concentration approximatingwhole serum. For comparison with the auxo-trophic derivative C5P, the streptomycin-sus-ceptible wild-type C5 was also tested. Figure 1shows the growth curves and the calculateddoubling times. Growth in 10% mouse serum(in phosphate buffer) was slower and the finalyield less than in 90% serum for all four Sal-monella strains. The histidine-requiring C5P,though mouse-virulent, did not grow well inmouse serum (decrease in growth rate whenviable count reached about 108), probably becauseof limitation of histidine in mouse serum (onaddition of histidine, C5P grew normally).The doubling times in 90% serum were: C5P(streptomycin-sensitive, histidine-requiring, viru-lent), 33 min (in serum supplemented withhistidine); C5 (streptomycin-sensitive, proto-trophic, virulent), 27 min; C5R (streptomycin-resistant, prototrophic, nonvirulent), 34 min;SW1444 (streptomycin-resistant, methionine-,phenylalanine-, and tyrosine-requiring, non-virulent), 43 min.

Rate of clearance from the blood in normalmice. Soon after intravenous inoculation ofcertain nonvirulent Salmonella into normal mice,the bacteria are rapidly removed from the circu-lation, whereas virulent Salmonella are onlyslowly cleared (3, 9). The rates of clearance of therecombinant and parental Salmonella strainswere therefore tested in normal mice. It was ex-pected that the nonvirulent recombinant C5Rand the nonvirulent parent SW1444, but not thevirulent parent C5P, would be rapidly phagocy-tized. Figure 2 shows the clearance of these

TABLE 2. Pattern of resistance to phagesa

Smooth-specific phages Smooth- and Rough-specific phagesStrain rough-specific

phageP22 P22h 9Na (Felix 0) 6SR Br2 Ffm Br6O P221 C21

S5W444 + + + + - - -C5 + + + + - - - - - -C5P + + + + - - - - - -M206b - - + + - - + + + -C5R + + + + - - - - - -Lister - | + + |

a Nutrient agar plates were surface-inoculated and spotted with 0.01 ml of phage lysate (108 plaque-forming units/ml). The plates were incubated at 37 C and examined at 5 and 18 hr (B. A. D. Stocker,personal communication; 18). Symbols: + = lysis; - = no lysis.

b The M206 strain that was used in clearance tests (Fig. 2) was no longer available. The strain shownwas a culture subsequently provided by D. Rowley.

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1346KRISHNAPILLAI AND KARTHIGASU

Salmonella strains during the first 5inoculation (determined by viable eblood samples), and the calculated "Vindices" (K). As expected, the nonv

HOURS HOURS

FIG. 1. Growth curves ofSalmonella in mlat 37 C. The time (minutes) refers to douSymbols: X, sirain C5 in 90% serum; 0, s510% serum; 0, strain C5P in 90% seru

mented with 20 ug of histidine per ml); A,

in 90% serum; A, strain C5P in 10% serum

SW1444 in 90% serum; O, strain SWJ44serum; *, strain CSR in 90% serum; O, sin 10% serum.

7.7

~~~~~~~~~~Lister

§ 7.U. ~~~~~~C5R

0

C5, 7*3LU ~~~~~~C5P

0

ED 6+z SW1444M %,/M206

MINUTES

FIG. 2. Rate of intravenous clearance of 5

Symbols: A, strain Lister; 0, strain CSR;C5; *, strain C5P; A, strain SW1444;M206.

min after abony parent SW1444, though smooth, wascounts of rapidly cleared (K = 0.137), whereas the virulent)hagocytic parent C5P was not (K = 0.015). However, con-irulent S. trary to expectation, the nonvirulent recombinant

C5R was not rapidly cleared (K = 0.007). Beforethe significance of this result could be assessed,

Jn(0-2-5h two points had to be considered. First, it wasin(0258hr) possible that the mice used in these tests were(205-8 " somehow different from mice used by other in-(4"-8' vestigators. Second, the rate of clearance deter-

mined by enumeration of viable bacteria maydiffer from those determined by using the 32plabel technique described by Jenkin (8). Therates of clearance of the S. typhimurium strainsC5 and M206, which had previously been char-

in acterized by 32p label-monitoring in other mice(8), were therefore examined with the mice used

ni in these studies by enumeration of viable bac-teria (Fig. 2). The nonvirulent strain M206 wasrapidly cleared (K = 0.163) and the virulentstrain C5 was only slowly cleared (K = 0.010).These K values closely resemble those of Jenkin(8). We conclude that clearance experiments ascarried out by us give the same kind of results as32p label experiments. The result with C5R there-

ouse serum fore indicates that some nonvirulent Salmonellabling time. strains are not rapidly cleared. It was thought oftrain CS in interest to see how another Salmonella strain,m (supple- identical to the recombinant C5R in its Kauff-strain C5P mann-White antigens (1, 4, 5, 12: b * 1, 2),v Q, strain and like it of low virulence (LD50 = 101 bacteria),train C50 would fare in the circulation of normal mice.

The clearance of such a strain (S. paratyphi Bstrain Lister) is shown in Fig. 2; like C5R, itwas very slowly cleared (K = 0.010).

K Thus, the smooth, nonvirulent Salmonella0*010±0-018 (S. abony SW1444) was rapidly cleared even

though it does not obviously differ in 0-somaticantigens from a virulent Salmonella which is not

0.007±0018 cleared (S. typhimurium C5P) (8, 9). Some non-

0.010±-0012 virulent smooth strains (recombinant C5R andS. paratyphi B Lister) are not rapidly cleared,

0.015±0012 which implies that susceptibility to clearance isnot the only cause of nonvirulence in smoothSalmonella.

Effect of antibody on rate of clearance. Theslow clearance of some bacteria from the mousecirculation results from absence of antibody(1, 8). The slow elimination of C5P and C5R

0137+0.043 might therefore be due to the absence of anti-01630036 body directed against these strains in the serum

of normal mice. To test this, the clearance innormal mice of bacteria opsonized by priorexposure to immune mouse serum was examined

ialmonella. (Table 3). For comparative purposes, the K*, strain values (from Fig. 2) of nonopsonized bacteria0, strain were also included. Untreated cells of strains

C5P and C5R were scarcely cleared in 5 miin

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TABLE 3. Effect of antibody on intravenous clearance of bacteria

Normal mice

Strain Normal micea Immune mice(nonopsonized) (nonopsonized) Opsonized with C5R Opsonized with SW1444immune serum immune serum

C5P 0.015 0.012b 0.140 i 0.030 0.178 i 0.024 0.244 i 0.026C5R 0.007 0.018 C 0.158 + 0.046 0.189 0.040SW1444 0.137 4 0.043 0.180 0.046

a The K values are from Fig. 2 for comparison.b "Phagocytic index" (K).C Not done.

(K = 0.015 and 0.007, respectively; Fig. 2), butthey were very rapidly cleared (K for C5P =0.178 - 0.244, and for C5R = 0.158 - 0.189)if they had been exposed, prior to inoculation, toserum from mice immunized 2 weeks previouslyeither with C5R or with SW1444. C5P was alsorapidly cleared from the circulation of micewhich had previously been actively immunizedagainst C5R (K = 0.140). The rapid clearanceof C5P and C5R after opsonization with immuneserum prepared against C5R or SW1444 can beexplained in terms of the identity of the Kauff-mann-White 0 antigens of strains C5P, C5R, andSW1444. These results indicate that the non-clearance of C5P and C5R in normal mice isprobably due to absence of sufficient opsonizingantibody in their sera.

Rate of intravenous clearance determined over30 min. Strains C5P and C5R were not clearedappreciably from the mouse's circulation in the5-min test (Fig. 2), and so clearance was ex-amined over a longer period (Fig. 3). The ratesof clearance of C5P and C5R differed only slightlyfrom those obtained over the 5-min period(C5P, K = 0.015 for 5 min and 0.015 for 30 min;C5R, K = 0.007 for 5 min and 0.016 for 30min). With both strains, however, only about30% of the bacteria remained in the mouse'scirculation at 30 min. In contrast, 99% of thebacteria of the nonvirulent parent SW1444 hadbeen cleared by 30 min. (Two K values forSW1444 are recorded, since the rate decreasedafter 10 min.)

Blood counts of recombinant and parentalSalmonella over a long period of time. As theclearance of the virulent parent C5P and thenonvirulent recombinant C5R did not differ overa 30-min period, viable counts were made over aconsiderably longer period (Fig. 4). The geo-metric mean of counts from five mice at eachtime period are shown. For all three Salmonellastrains, the number of viable bacteria per milli-liter of blood decreased in the first few hoursafter introduction. Even strains C5P and C5R,

MINUTESFIG. 3. Rate of intravenous clearance in 30 min.

Symbols: A, strain CSP; 0, strain C5R; 0, strainSW1444.

which were scarcely phagocytized initially (Fig. 2and 3), slowly disappeared from the circulation,with less than 5% remaining 2 hr after challenge.The decrease in blood viable count terminatedby 6 hr. From this time onwards, the behaviorof the three Salmonella strains was quite different.The virulent parent C5P rapidly increased innumbers in the blood till it reached 107 bacteria/ml, 30 to 36 hr after challenge, by which timenearly all the remaining challenged mice died.In the case of the nonvirulent recombinant C5R,there was little change in the blood viable counttill 48 to 72 hr. In mice challenged with the non-virulent donor parent SW1444, the bacterialpopulation in the blood did not increase ap-preciably. While these experiments were inprogress, groups of mice (15 per strain) thatwere similarly challenged were set aside to deter-mine cumulative mortality (Fig. 4). The period

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1348KRISHNAPILLAI AND KARTHIGASU

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FIG. 4. Distribution of Salmonella in the blood afterintravenous challenge with 5 X 107 to 10 X 107 bac-teria/mouse. Five mice were analyzed at each time.Cumulative mouse mortality at different times afterchallenge with the same dose are also shown. Symbols:A, strain C5P; 0, strain C5R; 5, strain SW1444.The vertical bars represent the range of counts aboutthe geometric mean.

of maximum mortality coincided with the ap-pearance of large numbers of bacteria in theblood.To determine whether the absolute number of

bacteria inoculated intravenously affected sub-sequent events in the blood, mice were inoculatedwith 5 x 105 to 10 x 105 bacteria/mouse (i.e.,with 5 X 104 to 10- X 104 LD5o of C5P, 0.05 to 0.1LD5o of C5R, or with 0.01 to 0.02 LD5o of SW1444)instead of with 5 X 107 to 10 X 107 bacteria/mouse (as in Fig. 4). The results (Fig. 5) showedthat all three Salmonella strains were removedfrom the circulation by 24 hr (count reducedto <1% of initial level), but by 48 hr there wasa divergence in the behavior of the strains. Inmice inoculated with the virulent strain C5P,the numbers of viable bacteria in the blood in-creased rapidly, by 72 hr exceeding 106 bacteria/ml, and all the mice died within 96 hr of inocula-tion. In mice inoculated with the nonvirulentC5R, small numbers of bacteria were recover-able till 72 hr, after which time blood sampleswere sterile (<20 bacteria/ml) till the end of theexperiment (day 13). Most mice challenged withthe nonvirulent SW1444 gave sterile blood cul-tures after 24 hr.

Recovery of viable bacteria of the recombinantand parental Salmonella in the liver and spleen. Thephagocytic cells of the reticuloendothelial system(RES) in the liver and the spleen are principallyresponsible for the removal of bacteria from theblood (1, 2). It was therefore of interest to studythe counts of viable bacteria in the liver and spleenof intravenously inoculated mice (Fig. 6 and 7).

Fio. 5. Distribution of Salmonella in the blood afterintravenous challenge with 5 X 105 to 10 X 105 bac-teria/mouse. Five mice were analyzed at each time.Symbols: A, strain C5P; 0, strain C5R; O, strainS W1444. The vertical bars represent the range of countsabout the geometric mean. Values below the axis lineindicate that the blood sample was sterile (no viablebacteria in 0.05-ml blood sample plated), and the num-bers within the symbols indicate the numbers of suchmice.

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FIo. 6. Distribution of Salmonella in the liver afterintravenous challenge with S X 105 to 10 X 10O bac-teria/mouse. Three mice were analyzed at each time.Symbols: A, strain CSP; 0, strain CSR; 0, strainSW1444. The vertical bars represent the range ofcounts about the geometric mean. The values below theaxis line indicate that the livers when cultuired yielded<500 bacteria/liver, and the numbers within the sym-bols indicate the numbers of such mice.

The mice were inoculated with 5 X 105 to 10 X105 bacteria/mouse. For each bacterial strain,three mice were killed per time. The zero-timevalues refer to counts on the organs of micekilled immediately after challenge. The number ofviable bacteria of the virulent strain C5P re-

covered from these organs increased, and by 72hr the bacterial population reached 108 to 109 perorgan. All of the remaining mice challenged withthe virulent strain died during the next 24 hr.The nonvirulent recombinant C5R and the

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FIG. 7. Distribution ofSalmonella in the spleen afterintravenous challenge with 5 X 10' to 10 X 105 bac-teria/mouse. Three mice were analyzed at each time.Symbols: A, strain CSP; 0, strain CSR; 0, strainSW1444. The vertical bars represent the range ofcounts about the geometric mean. The values below theaxis line indicate that the spleens when cultured yielded< 100 bacteria/spleen, and the numbers within the sym-bols indicate the numbers of such mice.

nonvirulent parent SW1444 failed to increase innumber in the liver. A similar situation prevailedin the spleen except for a small increase in thefirst 24 hr after inoculation. The recombinantC5R appeared to fare a little better than SW1444between days 1 and 4 in the spleen. Both non-virulent strains were present in low numbers inthe liver (<500 bacteria/liver) by the 10th day,and in the spleen by the 16th day ( < 100 bacteria/spleen). The significant point is that, whenequivalent numbers of bacteria were introducedintravenously, the virulent strain C5P had mul-tiplied within 72 hr to population levels 1,000-foldgreater than the nonvirulent strains.

DISCUSSIONThe lack of mouse virulence of certain strains

of, for instance, S. typhimurium, results fromknown causes, such as a requirement for growthfactors (e.g., adenine) which are absent frommouse tissue (6), or from roughness which isknown to determine extreme susceptibility tophagocytosis (4). In other cases, the cause oflack of virulence is not apparent. An earlierinvestigation (11) concerned the genetic determi-nation of the nonvirulence (LD5O = 108 bacteriaby intraperitoneal inoculation) of a strain of S.abony which is smooth and which has an 0-

antigenic formula identical with that of a mousevirulent strain of S. typhimurium (LD5o = <50bacteria). In crosses of an Hfr derivative of thisS. abony strain with the mouse virulent F S.typhimurium, the degree of virulence of re-combinants suggested that the S. abony straincarried "low-virulence" genes in two regions ofthe chromosome. The present report describes

experiments designed to allow examination ofthe physiological basis of nonvirulence of theS. abony strain, by comparing the behavior invarious tests of the two parental strains and arecombinant which was as nonvirulent as the S.abony donor.The results of phage sensitivity tests showed

that the nonvirulent S. abony parent SW1444and the nonvirulent recombinant C5R were fullysmooth, like the virulent S. typhimurium parentC5P (Table 2). All three strains were also re-sistant to the bactericidal action of normalhuman serum (unpublished data); results of thistest have been interpreted as indicating a possiblecorrelation between mouse virulence and in-susceptibility to human serum (16). In contrastto the three strains mentioned above, the non-virulent S. typhimurium strain M206 is sensitiveto certain rough-specific phages (Table 2), andboth this strain and S. paratyphi B Lister arevery sensitive to killing by human serum (un-published data); these results suggest possiblecauses of their nonvirulence.Hobson (7) found that mutants of S. typhi-

murium which are highly resistant to strepto-mycin grew more slowly in broth than did thestreptomycin-susceptible ancestor, and the mu-tants were of diminished virulence. Growthcurves determined in 90% normal mouse serum(Fig. 1) showed that the two streptomycin-re-sistant strains tested (SW1444 and C5R), bothnonvirulent, had longer doubling times than didthe streptomycin-sensitive virulent strain C5(the wild-type ancestor of C5P). These resultsare in agreement with those of Hobson (7). Thegrowth rate of the virulent strain C5P was low,even in serum supplemented with histidine (thegrowth requirement of C5P), but it is possiblethat with a larger supplement of histidine itwould have doubled faster. In any case, the non-virulence of the recombinant C5R was unlikelyto be due to inheritance of the streptomycin-resistance allele from the S. abony SW1444 donor,since some recombinants which inherited thesame allele from SW1444 in crosses with anothervirulent subline of S. typhimurium C5 were stillvirulent (11).When bacteria are injected intravenously into

normal mice, cells of strains which are nonvirulent(to the mouse) are in general rapidly removedfrom the circulation by phagocytosis, whereascells of mouse-virulent strains are much lessrapidly cleared. This has been shown with S.typhimurium (3, 8), S. gallinarum (9), and withKlebsiella pneumoniae (9). Presumably, sus-ceptibility to rapid clearance by phagocytosis iseither the cause of the diminished virulence of the

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KRISHNAPILLAI AND KARTHIGASU

strains concerned, or it results in some way fromthe character, e.g., roughness, which causes theirlow virulence. But not all strains of low virulencefor the mouse are rapidly cleared, because thereare strains (Vi-negative) of S. typhi (5), S.adelaide (5, 10), Escherichia coli 011OB4 (1),and S. paratyphi B (this paper) which are smoothand yet poorly phagocytized. These exceptionshave been noted previously (3, 5).

In the present study, clearance of intravenouslyinoculated viable bacteria was followed by mak-ing counts of viable bacteria in blood samplestaken at short intervals after injection. Whenstrains whose clearance in normal mice had beeninvestigated by Jenkin (8) were tested in this way,the rates of clearance ("phagocytic indexes")determined from viable counts were essentiallysimilar to those determined from clearance ofradioactivity of intravenously inoculated viablebacteria labeled with "P (8; Fig. 2). We inferthat the clearance (in terms of viable count)which we observed results, mainly or entirely,from removal by phagocytosis by fixed cells ofthe RES (liver and spleen mainly), rather thanfrom killing in the bloodstream (e.g., phago-cytosis by circulating leukocytes), and we havetherefore calculated "phagocytic indexes." Earlierwork cited above suggests that all Salmonellastrains susceptible to rapid clearance are of lowvirulence, but that some (smooth) nonvirulentstrains are not rapidly cleared. In the presentwork, the mouse-virulent (smooth) S. typhi-murium parent strain C5P was, as expected,cleared very slowly in normal mice (K = 0.015;Fig. 2). The relatively nonvirulent S. abonyparent SW1444, though smooth by the criteriaused, was rapidly cleared (K = 0.137; Fig. 2)from such mice. Testing the virulence of re-combinants from crosses of these two strainsindicated that the low virulence of the S. abonyparent (LD6o = 108 bacteria) was determined bythe joint effect of two loci (chromosomal regions),one between inl and metA and the other betweenstrA and aroD (11, 14, 17). The presence of oneonly of these "low-virulence" loci in a recom-binant results in intermediate virulence (LDOn =10' bacteria), as shown previously (11). Thenonvirulent S. abony Hfr strain SW1444 is sus-ceptible to rapid clearance, and it might thereforehave been expected that one of the two "low-virulence" loci determined the susceptibility torapid clearance of strain SW1444 and thus itsdiminished virulence. However, the smoothrecombinant CSR, obtained from a backcross insuch a way that it had both the S. abony "low-virulence" loci in an S. typhimurium geneticbackground, was as nonvirulent as the S. abonyparent (LDIo = 108 bacteria; 11), but was no more

rapidly cleared (K = 0.007; Fig. 2) than thevirulent S. typhimurium parent C5P. It thereforeappears that both of the "low-virulence" locidetermine diminished virulence by mechanismsother than susceptibility to rapid clearance. Thephysiological basis and genetic determination ofthe susceptibility of SW1444 to phagocytosisremain unknown. It is therefore not yet feasibleto test whether this character would by itselfaffect virulence (e.g., in a recombinant lacking thetwo mapped "low-virulence" loci). The slowclearance of the recombinant C5R presumablyreflects inefficient phagocytosis (owing to limi-tation of antibody), since cells of C5R becomesusceptible to rapid clearance upon opsonizationwith immune serum (Table 3).

Biozzi and his colleagues (3) reported that anonvirulent E. coli-S. typhimurium recombinantwas rapidly cleared from the blood, like itsnonvirulent E. coli donor parent and unlike itsvirulent S. typhimurium recipient parent. Al-though the recombinant possessed the charac-teristic somatic and flagellar antigens of thevirulent parent, minor differences in the surfaceantigens of such a recombinant and the recom-binant C5R (described here) may account for thedifference in behavior of the two types of re-combinants in clearance tests.The nonvirulent recombinant C5R and the

virulent C5P were both poorly phagocytizedinitially in normal mice (Fig. 2 and 3), and wetherefore made a study of later events in thehope of discovering which of these events wererelated to the final outcome of infection. Whenintravenous challenge doses were 5 X 107 to 10 x107 viable bacteria, both C5P and C5R wereslowly cleared from the mouse circulation but re-emerged in the bloodstream after a variabletime (Fig. 4). The re-emergence was characteristic(i.e., occurred earlier with the virulent C5Pthan with the nonvirulent C5R), even thoughboth of the strains finally killed their hosts. How-ever, if the challenge dose was 5 X 10' to 10 x105 bacteria, a different situation developed(Fig. 5). The numbers of virulent C5P in the bloodincreased and the challenged mice died, but bothof these events occurred later than in the experi-ments with the larger dose. There was no re-surgence of the nonvirulent C5R, no bacteriawere detectable in the blood 5 to 7 days afterchallenge (Fig. 5), and all of the mice survived.This characteristic of a smaller dose level haspreviously been observed in the Salmonella-mouse infection system (7).

Prior to death of mice challenged intraperi-toneally with small numbers of virulent S.typhimurium, the bacterial population in theorgans (associated with bacteremia) amounts to

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NONVIRULENT SALMONELLA RECOMBINANT

108 to 109 bacteria (7). This was confirmed herein the case of mice dying from challenge with thevirulent parent strain C5P (Fig. 5-7). It maytherefore be asssumed that death of animalsinfected with the virulent S. typhimurium wasassociated with marked bacterial proliferation.On the other hand, the nonvirulent recombinantC5R and the nonvirulent parent SW1444 failedto proliferate markedly (Fig. 5-7). Similar ob-servations have been made with a relatively non-virulent, streptomycin- resistant mutant of S.typhimurium (7).One may conclude that the recombinant C5R

lacks the ability to proliferate in the tissues of themouse, presumably because it possesses one orboth of the two "low-virulence" loci of its non-virulent S. abony parent SW1444. This inabilityto proliferate is not related to nutritional re-quirement, because C5R is prototrophic, is notassociated with loss of the smooth character (asdetermined in phage sensitivity tests), and doesnot result from susceptibility to rapid intra-venous clearance.

ACKNOWLEDGMENTS

We are grateful to B. A. D. Stocker, R. J. Roantree, andD. G. MacPhee for helpful criticism during the preparation of themanuscript, and Othman bin Mohamed and M. Manivelu fortechnical assistance. We thank P. H. Makela, D. Rowley, andR. Bhagwan Singh for the gift of bacterial strains. We also thankJ. Dreyer for performing the phage pattern tests.

LITERATURE CITED

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16. Roantree, R. J., and J. P. Steward. 1965. Mutations to penicil-lin resistance in the Enterobacteriaceae that affect sensi-tivity to serum and virulence for the mouse. J. Bacteriol.89:630-639.

17. Sanderson, K. E. 1967. Revised linkage map of Salmonellatyphimurium. Bacteriol. Rev. 31:354-372.

18. Wilkinson, R. G., and B. A. D. Stocker. 1968. Genetics andcultural properties of mutants of Salmonella typhimuriumlacking glucosyl or galactosyl lipopolysaccharide trans-ferases. Nature 217:955-957.

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