INFEcTiON AND IMMUNITY, Jan. 1975, p. 180-192 Copyright i 1975 American Society for Microbiology

Vol. 11, No. 1 Printed in U.S.A.

Variations in Properties of L-Forms of Pseudomonas aeruginosa ROBERT BERTOLANI, SANFORD S. ELBERG, AND DORIS RALSTON School of Public Health, University of California, Berkeley, California 94720 Received for publication 15 August 1974

In a study of the pathogenic potentials of Pseudomonas L-forms, three unstable L-forms were derived by carbenicillin induction from a mouse virulent strain of Pseudomonas aeruginosa, Rosenthal 180. One L-form, induced on a sucrose-stabilized medium, grew more slowly and differed in a number of properties from two other L-forms induced on a medium supported with polyvinylpyrilidone. After adaptation to a common liquid medium, the three L-forms differed with respect to colonial shape on solid medium, growth rate, certain biochemical properties, antibiotic sensitivities and antigenic surface, and virulence for mice. The L-form may revert in vitro to a serotype different from that of the parent culture. The revertant may acquire new antibiotic resistances and sensitivities in the absence of previous exposure to the drugs and enhanced resistance to the L-inducing agent. The three L-forms showed a characteristically lower, but wide, range of virulence than did the parental form. Though death of mice was accompanied by reversion of the L-forms in vivo to the bacterial form, reversion in vivo was not necessary for virulence of L-forms. Modification of residual cell wall antigens accompanied the induction of each L-form as determined by type-specific antisera.

Pseudomonas aeruginosa is an opportunistic pathogen causing severe, often fatal, infections in patients with certain underlying diseases. Two symposia (19, 20) have recently dealt with the efficacy of carbenicillin (CB) for treatment of many types of gram-negative infections, especially those involving septicemia and bacteriuria from P. aeruginosa. The current widespread use of CB for treatment of P. aeruginosa infectiQns and the recent demonstration by Hubert et al. (9) of in vitro induction of L-forms of P. aeruginosa by CB prompted us to study the potential of L-forms to infect and survive in vivo. The subject of L-form pathogenicity has recently been reviewed (4). Three distinctly different L-forms from a single strain of P. aeruginosa were studied in terms of their induction and their various properties. MATERIALS AND METHODS Bacterial strains. P. aeruginosa, Rosenthal 180 strain (ATCC 19660), derived from a case of human septicemia and capable of causing septicemia in mice, was used for induction of L-forms. The strain was maintained on nutrient agar (Difco) slants and was transferred every 2 months. It was stored at 4 C. Other strains used included La Page (ATCC 23389), Jones 452 (ATCC 19142), and Jones 822 (ATCC 19144). Media. Brain heart infusion (Difco) broth (BHI) was used throughout as the basal medium, supplemented with 4% (wt/vol) polyvinylpyrilidone (PVP;

Plasdone C, GAF Corp., New York, N.Y.) (11) or 0.5 M sucrose as osmotic stabilizer for induction (11). Two percent (wt/vol) bovine serum albumin (Armour Pharmaceutical Co., Chicago, Ill.) and 0.001% (wt/ vol) MgSO4 were incorporated for L-form induction and propagation (9). For solid miedium, 1% (wt/vol) agar (Difco) was added to BHI broth. All media were prepared with double distilled, deionized water. Induction. Carbenicillin (Beecham Pharmaceuticals, Clifton, N.J.) was used for induction of the L-form. Three methods of induction were used. (i) CB gradient in agar, sucrose stabilizer; slow adaptation to broth. A saline suspension of 18-h Trypticase soy agar (TSA) growth of the parent bacterium was spread on a gradient (0 to 300 Ag/ml) CB plate with 0.5 M sucrose as osmotic stabilizer. When growth appeared, an agar block containing several colonies was cut out and pushed across another plate containing CB (300 ug/ml). Subsequent subcultures were made to medium containing increasing concentrations of CB. By the 69th passage, the L-colonies on medium with 5,000 gg of CB per ml (sucrose L-medium) still reverted upon subculture to antibiotic-free sucrose medium. To adapt to a medium with 4% PVP (P4 agar) in place of sucrose as osmotic stabilizer, L-colonies were picked from sucrose L-medium into P4-L broth (5,000 ,g of CB per ml), and after incubation for 48 h, portion was streaked onto P4-L agar. L-colonies arose, which were serially passed through decreasing concentrations of agar containing 5,000 jAg of CB per ml and finally into P4-L broth. The culture grew slowly at first, producing a noticeable turbidity after 14 days of incubation, when the second broth transfer was made. Subse-

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quent broth transfers were made every 4 to 6 days in increased concentration (8,000 jg/ml) of CB. This culture was designated b, (slow grower). (ii) CB (6,500 jig/ml) in BEIl broth, no added stabilizer. From an 18-h TSA culture of the parent bacillus, cells were inoculated into BHI broth containing 6,500 jg of CB per ml. After a few days of incubation, the culture showing predominantly granular elements was streaked onto P4-L agar (9,000 jug of CB per ml). Typical fried-egg colonies consisting of coccoid and granular forms digging into the agar arose. These forms were adapted to grow in P4-L broth through decreasing concentrations of agar. At first, growth could not be transferred from 0.9 to 0.8% agar at 9,000 jig of CB per ml; the concentration of CB was lowered to 6,500 jig/ml to facilitate transfer from 0.9 through 0.5% agar, after which rapid subculture through lower agar concentrations and consequently to broth was possible at 8,000 jig of CB per ml. Turbidity in L-broth appeared after 7 days of incubation. Subcultures grew more rapidly than the culture isolated by the sucrose-gradient CB technique and was designated L, (fast grower). (iii) Four percent PVP as osmotic stabilizer, 8,000 jg of CB per ml; direct transfer to broth. Preliminary studies indicated that 8,000 jig of CB per ml incorporated into P4 agar yielded a high frequency (3%) of induction compared with that observed on agar containing 1,000 jg of CB per ml (0.0005%). Stationary P4 broth-grown culture of the parent bacterium was spread onto the surface of P4-L agar (8,000 jg of CB per ml). Typical fried-egg colonies arose after a few days of incubation. These were passed once on P4-L agar and then transferred directly into P4-L broth and incubated in a reciprocating shaker at 130 strokes per min in a water bath at 37 C. Growth was apparent after 24 h as large coccoid bodies. These L-phase bodies transferred easily in P4-L broth (8,000 jg of CB per ml). The culture was termed Ld to indicate its direct passage into broth, in contrast to the slow broth adaptation of b, and Lb. Maintenance and harvest of L-phase cultures for tests. L-form stock cultures were routinely transferred every 3 to 6 days in 5 ml of P4-L broth (8,000 jig of CB per ml) in screw-cap tubes (130 by 20 mm). Cultures were incubated on the reciprocating shaker for 24 h and stored at 4 C until the next transfer. L-phase organisms for experimental uses were cultured in 25 ml of P4-L broth in 250-ml Erlenmeyer flasks equipped with a side arm to fit a Klett-Summerson colorimeter for direct reading of turbidity at 660 nm. Flasks were incubated on the shaker until a stationary phase had been reached. At early stationary phase, cultures were spun at 4 C for 10 min at 3,020 x g in a Sorvall refrigerated automatic centrifuge and resuspended in phosphate-buffered saline with 4% PVP at pH 7.2. Antibiotic sensitivity. Suspensions of L-form cultures were spread onto the surface of P4-L agar. Antibiotic disks (Difco) (5) were then positioned on the agar surface and diameters of inhibition were recorded after 24 h of incubation. Revertants and parent strain were grown and tested in the absence of CB.

181

Antisera. Type-specific antisera (6) to somatic antigens of seven types of P. aeruginosa were kindly supplied by George Cole of Parke, Davis & Co., Detroit, Mich. In addition, antisera to Rosenthal 180 strain and two other strains of P. aeruginosa were prepared in our laboratory. Saline suspensions of overnight growth of strains on TSA were steamed at 100 C for 2.5 h, washed four times and resuspended to a final turbidity equivalent to 1011 viable cells/ml. New Zealand white rabbits (4 to 4.5 lb [about 1.8 to 2.0 kg]) were injected subcutaneously with 1 ml of the cell suspension diluted 1:1 in complete Freund adjuvant. Sixty days later rabbits were given a first booster intravenously, followed in 3 weeks by a second intravenous injection. Rabbits were bled from the heart 4 to 5 days later. After clotting the serum was decanted, spun, and sterilized by filtration through a 0.45-jim filter (Millipore Corp., Bedford, Mass.). Serum was stored at 4 C. Agglutinations. Slide agglutinations were performed with Parke Davis typing sera and with other pseudomonas antisera prepared as described above on PBSP4 suspensions of L-form cultures harvested in the stationary phase. A loop delivering approximately 0.01 ml of each antiserum diluted 1:2.5 in saline was added to a loop of cell suspension on glass slides. Evidence of clumping was observed under a dissecting microscope at low power. All clumping occurred within 5 min at room temperature. Control suspensions were prepared with normal rabbit serum and with PBSP4. Because the L-forms were induced from the parent on different occasions, serological differences were not, regrettably, compared at the same passage number. Though increased passage may have effected serological changes, bd typed from passages 6 through 30 retained antigens 3 and 7 of the parent organism. Lb culture had already changed serologically from the parental type as early as passage 32. Biochemical tests. Tests for sugar utilization were performed in OF basal medium (Difco); N Taxo disks (BBL) were used for oxidase determination; all other tests were performed with Difco media according to procedures prescribed in the Difco Manual (5). Lform cultures were tested in media supplemented with 4% PVP and to which 8,000 jg of CB per ml was added. Phage lysogenicity. An indicator strain of P. aeruginosa, Jones 822, for the lysogenic phage of the Jones 452 strain was found to be sensitive to lysis by a phage lysogenic for the parent bacillus. To test for lysogenicity of the L-forms and revertants, TSA plates were swabbed with a standard suspension (approximately 10, organisms per ml) of the indicator strain immediately before spotting of serial 10-fold dilutions in PBSP4 buffer of each culture to be tested. Plates were incubated overnight before being observed for discrete plaque information. Test for mucopeptide and residual cell wall material. The presence of residual cell wall components on the L-form was tested for by treatment of the cells with ethylenediaminetetraacetic acid (EDTA), shown to solubilize cell wall components of gram-negative organisms (7). Test for mucopeptide was carried

182

BERTOLANI, ELBERG, AND RALSTON

out by treatment with lysozyme in EDTA in tris(hydroxymethyl)aminomethane (Tris) buffer (16). Stationary-phase growths of parent and L, cultures were spun and washed twice in 0.3 M Tris-hydrochloride buffer, pH 8.6. The buffer was supplemented with 0.25 M sucrose. For washing, 0.1 M Mg2+ was incorporated to prevent lysis and clumping of L-forms. After the final wash, parent and Ld cultures were resuspended in sucrose-stabilized buffer without Mg2+ to a final turbidity of approximately 90 units on a KlettSummerson colorimeter. Five milliliters of each cell suspension was added to a series of colorimeter tubes containing either (i) 0.05 ml of 4% (wt/vol) EDTA plus 0.03 ml of 2% (wt/vol) lysozyme, (ii) EDTA alone, or (iii) lysozyme alone. Control tubes contained 0.08 ml of deionized water. Tubes were incubated at 37 C, and the drop in turbidity was read on the colorimeter. Virulence tests. Virulence of the three L-form cultures was tested in 8-week-old male albino mice (Namru strain), obtained from the Naval Biomedical Research Laboratory in Oakland, Calif. Mice were housed in plastic cages equipped with water bottles and were fed Purina mouse chow ad libitum. L-form cultures were harvested in early stationary phase in PBSP4 for injection into mice. Mice were injected with 0.5 ml of the appropriate cell culture intraperitoneally. After all injections were complete, 0.05 ml of the dilutions was spread onto the surface of P4-L agar to determine the minimal number of colony-forming units (CFU) injected per mouse. To recover L-phase organisms surviving in mice, organs were removed and homogenates were made by grinding the organs with mortars and pestle in the presence of P4 broth. Portions of organ homogenates and of peritoneal fluid and blood were spread onto the surface of P4 agar with and without CB (8,000 /Ag/ml). Virulence of the L-forms and parent was compared by injecting groups of six mice each with serial twofold dilutions of the appropriate inoculum. Whereas injection of 107 vegetative cells killed approximately 50% of the animals within 24 h, 4 x 107 to 8 x 107 CFU of the Ld culture was required to kill 50% of the animals within 24 to 48 h. Doses ranging from 2 x 108 to 4 x 108 CFU of L8 culture were needed to cause death of some animals. Generally, L8 tended to produce mild illness followed by recovery of the majority of mice. (Severe illness followed by death of the animal when injected with a lethal dose of Ld was characterized by the appearance of a crusty, yellowish exudate around the eyes, hunching and sluggishness of movements, paralysis of hind limbs, and eventual respiratory failure.) With doses equivalent to L8, L, produced no signs of illness or death. In studies where mice infected with Ld were given injections of different doses of CB intraperitoneally, mice were inoculated with approximately 2 x 108 CFU, sufficient to kill all mice usually within 24 to 36 h. In all experiments control groups not receiving CB were included.

INFECT. IMMUN.

form cultures, differing in colonial morphology, growth rate, ease of reversion, serology, osmotic lability, antibiotic spectrum, and virulence for mice (Fig. 1). Growth on agar. On P4-L agar the three L-form cultures exhibited distinct colonial differences (Fig. 2a-c). Lf and L8 colonies were small (0.5 to 1.0 mm) at 72 h, whereas Ld colonies developed faster and by 24 h were 1 to 2 mm. The minute central core of L8 and the almost imperceptible core of Ld contrasted with the intensely stained core of Lf. Also, the almost centerless appearance of the Ld colonies after propagation contrasted sharply with its appearance when first induced on P4-L agar from the parent (Fig. 2d). Microscopically, these intensely stained central cores were heavily granulated and imbedded in the agar. Grossly, Ld and Lf colonies were convex and translucent, whereas L8 colonies were flat or of low convexity and nearly transparent. Despite the colonial variation of the three L-form cultures, the morphological changes in individual L-phase bodies during incubation on solid medium were essentially the same. Microscopically, large and intermediate bodies predominated in early colonial development, giving way during continued incubation and increased colony size to smaller granular forms (Fig. 2e-g). After 7 days of incubation on P4-L agar, motile rods and filaments were observed at the periphery of some Ld colonies. Growth in broth. The differences in growth rate observed on agar became even more apparent in liquid medium. L8 was characterized by the longest lag phase (about 24 h versus 8 and 16 h). All three L-forms appeared in broth culture as clumps of L-phase elements, varying from granular forms to larger coccoid bodies of varying diameter (Fig. 3a). Larger coccoid bodies (3) not in clumps were more characteristic of Ld than of Lf or L. (Fig. 3b). At early stationary phase, fibrous clumps occurred in both Ld and Lf broth cultures; the onset of stationary phase in Ld was additionally marked by the sudden appearance of green pigment, characteristic of the parent culture. Ld also produced green pigment on P4-L agar after 36 h, but no parental morphological forms were evident. L8 began producing a melanin-like pigment at the onset of stationary phase. The production of pigment by unstable L-phase organisms is in contrast to the findings reported by Hubert et al. (9), whose L-form of P. aeruginosa failed to RESULTS produce any detectable pigment. However, they Three separate attempts at induction and induced a stable L-form from a different parenadaptation to broth yielded three unstable L- tal strain of P. aeruginosa.

L-FORMS OF P. AERUGINOSA

VOL. 11, 1975

183

Parent bacillus TSA Gradient CB, sucrose-stabilized agar

P4 broth (shake)

Inoculated BHI broth (unstabilized) containing 6,500 ug of CB per ml

P4-L agar (8,000 ug of CB per ml)

Serial passage on sucrose media while Streak after 48 h of incubation to P4 agar increasing CB to 5,000 ug/ml (69 pas- containing 9,000 ug of CB per ml sages)

P4-L broth (8,000 ug of CB per ml)

Incubate L-colonies on agar block Transfer L-colonies from 1.0% agar in from sucrose medium in P4 broth graded steps to 0.5% agar at 5,000 ug of containing 5,000 ug of CB per ml CB per ml

Ld

Streak from P4 broth containing su- Transfer from 0.5% agar through 0.1% agar crose agar block to P4-L agar (5,000 ug at 8,000 ug of CB per ml of CB per ml) Pass through decreasing agar concentrations of P4-L agar

Static P4 broth at 5,000 ug of CB

Static P4 broth at 8,000 ug of CB per ml

per ml

P4-L broth (8,000 ug of CB per ml) Shake culture

L.

Lf

FIG. 1. Methods of induction of L-form cultures.

CB dependence. The Ld and L8 cultures did not form L-colonies on antibiotic-free P4 agar; only revertant colonies developed. With continued passage of the Ld variant in P4-L broth, the proportion of the culture able to revert immediately upon plating on antibiotic-free P4 agar declined sharply (Table 1). Neither increased PVP concentration, substitution of 4% PVP with 0.5 M sucrose as osmotic stabilizer, variations in pH, nor the addition of Mg2+ to P4 agar enhanced the capacity of Ld and L cultures to revert or grow as L-colonies on antibiotic-free medium. (Earlier experiments with the Lf culture while it was being propagated in static broth culture were performed to determine what effect increased concentration of Mg2+ or su-

crose would have on reversion. Though no increase in the frequency of reversion occurred, we observed that the incorporation of 0.2% Mg2+ or 0.7 M sucrose into BHI agar favored the transition of coccoid bodies to filamentous and rod-shaped forms. The morphological transition to rods was not observed at 0.6% Mg2+; high Mg2+ preserved the coccoid morphology. Formation of rods was less apparent at 0.3 M than at 0.7 M sucrose.) Only the incorporation of CB into P4 agar (P4-L agar) allowed recovery of L-phase bodies on solid medium as L-colonies. Thus, with continued passage, the Ld culture became increasingly stable and/or dependent upon CB for initiation of L-phase growth on solid medium. The Ld culture grew as L-colonies

FIG. 2a-d. (a and b) Colonies of L, and LJ, respectively, after 72 h of incubation on P4-L agar (8,000 ,g of CB per ml). Note that L8 is more diffuse than Lf . Stained by the technique of Dienes. x 150. (c) Colony of Ld after only 24 h of incubation on P4-L agar (8,000 jg of CB per ml) showing its much larger size compared with L, and L,. Though unstained, its tiny core is evident. x100. (d) Colonies of Ld incubated for 48 h, when first induced from the parent on P4-L agar (8,000 ,gg of CB per ml); compare its size and morphology to Ld culture after several propagations on P4-L agar in (c). x 150. FIG. 2e-h. (e-g) Ld colonies at high magnification after 14, 24, and 36 h of incubation respectively. Note progression from large and intermediate-sized bodies to smaller more intensely stained granular elements. x 1,350. (h) L, colony on P4 agar after 48 h of incubation. Note that in absence of carbenicillin, faintly stained filamentous forms appear at periphery of more intensely stained central area of colony. x 1,350.

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185

186

BERTOLANI, ELBERG, AND RALSTON

INFECT. IMMUN.

I FIG. 3. (a) Clumps of L-phase elements in P4-L broth, 24 h. x1,350; (b) L-phase bodies growing singly in P4-L broth, 24 h. x 1,350.

on solid medium when the concentration of CB was lowered from 8,000 to 1,000 Ag/ml, but the size of Ld colonies was correspondingly reduced and the colony count dropped approximately 50%. The reasons for this CB dependence are not understood, but is not peculiar to Ld. LS behaved similarly in that it required CB for initiation of L-phase growth on solid medium and formed fewer colonies if the CB concentration was lowered, and its ability to revert immediately on antibiotic-free P4 agar was only manifested by a small portion of the culture. Similarly, initial induction from the parent bacterium was accompanied by a low frequency of induction to the L-phase on P4-L agar containing 1,000 ,ug of CB per ml (0.0005%), but a high induction frequency on P4-L agar containing 8,000 Ag of CB per ml (3%). Revertants

from Ld and L, (designated Rd and R,, respectively) acquired an enhanced ability to be induced into the L-phase; the frequency of induction of Rd on agar containing 1,000 and 8,000 Mg of CB per ml was greater than 10 and 50%, respectively. Unlike Ld and L", Lf showed increasing ability on passage to grow on antibiotic-free P4 agar (Table 1). The Lf colonies, after 36 to 48 h, first developed a dense center composed of granular growth in the agar, whereas surface growth was predominantly coccoid and intensely reactive to Dienes stain, except at the periphery of the colony, where long, filamentous transitional forms, faintly stained, were seen (Fig. 2h). With subsequent passage on P4 agar without CB, these colonies became less fried egg in appearance; granular forms disappeared and only large

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L-FORMS OF P. AERUGINOSA

coccoid and filamentous forms were observed. This "transitional morphology" maintained itself for nine passages on P4 agar. However, if passage was carried out on TSA, revertant colonies of typical, motile pseudomonas rods appeared after several subcultures. Osmotic lability. Deionized water as suspending fluid decreased colony counts of all three L-forms whether measured in the presence or absence of CB (Table 2). On CB medium, only revertant colonies developed. When L. was TABLE 1. Effect of continiued passage in carbenicillin on ability of L-form cultures to revert in absence of carbenicillin on stabilized medium

prepared in deionized water, a larger reduction occurred in number of CFUs capable of reverting on CB-free medium than on medium containing CB, indicating that some CFUs damaged with respect to reversion still retained the capacity to multiply as colonies on CB medium. An inductive medium was advantageous in recovering damaged L-phase bodies or walldefective forms, probably by forcing the cells to grow as L-colonies supported by the solid matrix of the agar medium. Serology. Ld and its revertant, Rd, retained the somatic antigens of the parent bacterium, but Ld differed from the parent bacillus and Rd

in its variable cross-reactivity with antisera prepared against two serologically unrelated Culture Passages Log differencesa strains of P. aeruginosa and with other typing sera (Table 3). In contrast to Ld and Rd, L, and 10 0.2 its revertant R& were strongly agglutinated by 20 0.5 three type-specific antisera, none of which ag24 2.4 glutinated the parent bacterium. Both L, and 25 2.6 26 3.2 Rf agglutinated strongly with antisera to the 30 5.3 unrelated test strains of P. aeruginosa, and agglutination reactions did not distinguish sur20 2.0 Lf face antigenic differences between Lf and Rf. 1.4 49 The L culture failed to agglutinate with any 64 0.8 of the antisera. The revertant, R., shared the 73 0.6 acquired antigenic determinants of Rf, as dem77 0.6 onstrated by its agglutination with typing sera 1 78 0.2 and 6, and of the parent, since it agglutinated with typing serum 7. R,, showed cross-reactivity 113 3.0 123 2.8 with two unrelated P. aeruginosa strains. The 3.4 126 agglutination of Rf and R. by antiserum J was aLog,0 CFU on carbenicillin medium minus log,0 expected because strain J was itself agCFU on medium lacking carbenicillin; number of glutinated by typing sera 1 and 6. Agglutination colonies on carbenicillin medium was relatively con- of Rf and R. by antiserum L (serotypes) suggests acquisition of additional determinants not destant for all determinations. TABLE 2. Osmotic lability of L-form cultures as determined by colony counts on media with and without carbenicillin L-forma

Ld L,

P4 agar' (x

Time in diluent

10')

Diluent

P4-L agarc (x 104) Diluent

CFU Ratio

( min)

Water

5 60

1.05 0.29

21,000

5 60

6.51 2.51

10,800

5 60

0.015 0.001

PBSP4

8,200

3,990 21.6 14.2

CFU Ratio

(PBSP4/water)d

Water

PBSP4

(PBSP4/water)

3.90 4.85

3.39 0.68

37,800 41,800

4.05 4.79

3.21 3.20

7.01 3.72

26,100

29,900

3.57 3.90

11,000 8,600

1.68 2.69

3.15 4.15

229 17.4

aFive-milliliter samples of stationary-phase growth of each L-culture were washed and resuspended in 5 ml of the appropriate diluent; serial 10-fold dilutions in each diluent were plated in duplicate. 'Medium lacking carbenicillin. c Medium containing carbenicillin. d CFU from PBSP4 diluent minus log10 CFU from water diluent. Figured as the

log10

188

BERTOLANI, ELBERG, AND RALSTON TABLE 3. Serotypes of L-form cultures, revertants, and parent Culture

Ld

Passage

Serotypea

6 13 29

3,6+,7,R,J+,L+ 3,4+,6+,7,R,J+,L± 3,4+,7,R

60 66 82

1,4,6,R,J,L 1,4,6,R,J,L 1,4+,6,R,J,L

109 116 130

None None Auto-agglutinating

Revertantsb ID

3

3,7,R 1,4,6,R,J,L 1,6,7,R,J,L

Rf R8

3,7,R

Parentc

a Strong agglutination is indicated except where the symbol + (= weak agglutination) appears. Numbers refer to Parke Davis typing sera, whereas letters R, J, L, refer to antisera prepared in our laboratory to P. aeruginosa strains Rosenthal 180 (parent), Jones 452, and La Page, respectively. hR,d, Revertant from 10th passage of Ld; R,, revertant from 32nd passage of Lf; R,, revertant from 92nd passage of L.. Revertants and parent grown for 24 h on TSA. c Typed identically when grown in P4 broth.

tected by typing sera 1 through 7. Antibiotic spectrum. The results of antibiotic sensitivity tests on the L-form cultures, revertants, and parent bacterium are shown in Table 4. Both Ld and L, showed a large increase in sensitivity to tetracycline, terramycin, rondomycin,

aureomycin,

and

declomycin. Ld

showed an increase in sensitivity to chloramphenicol and a slight increase in sensitivity to nalidixic acid (structural analogue of purine nucleosides) relative to the parent, whereas L, was much more sensitive to polymyxin than was Ld or the parent. Lf was highly sensitive to neomycin. In addition, Lf was slightly more resistant than the parent to aureomycin and declomycin. L, showed the greater increase in sensitivity to dihydrostreptomycin (DHSM) of the three Lforms. The antibiotics mentioned above, with the exception of nalidixic acid and polymyxin, primarily act at the level of protein synthesis (3). Why L, failed to show any increase in sensitivity to the antibiotics above, except neomycin, is not clear. The revertant of Ld, for the most part, re-

INFECT. IMMUN.

tumed to an antibiotic spectrum characteristic of the parent bacterium but retained some sensitivities of the L-form. Compared with the parent, the revertant, Rf, was more resistant to tetracycline, terramycin, chloramphenicol, aureomycin, and declomycin but more sensitive to DHSM, neomycin, kanamycin, and gentamicin. R, showed some similar acquired resistances, but since it was grown and tested on a different medium, comparison among Ra, its L-phase, and parent is not warranted. However, when the parent was grown and tested on TSA (as was Ra), there was no significant difference in its sensitivity to most of the antibiotics, in particular gentamicin, carbenicillin, and polymyxin. Therefore, we may state that Rd (and Rf) showed greatly enhanced sensitivity to gentamicin over the parent. Of all the revertants, only R, showed enhanced resistance to the L-inducing agent. It was resistant to up to 2,000 Ag of CB. The loss of sensitivity of Ra to polymyxin was expected since the antibiotic's main site of action is on the protoplasmic membrane (15). Acquisition of the cell wall by Ra would act as a barrier to the penetration of polymyxin. The enhanced sensitivity to kanamycin of Rf was not shared by other revertants of L-phases. Lr and Rf uniformly showed enhanced sensitivities only to the structurally related aminoglycosides: neomycin, kanamycin, DHSM, and gentamicin. Biochemical tests. The biochemical reactions of the L-form cultures and revertants were in general weaker and slower than for the parent (Table 5) except for Rd, which behaved identically to the parent. The test for nitrate reduction was terminated after 48 h, and the failure to detect nitrate reduction either to nitrite or to N2 by Ra and L8 may well reflect their slower growth. However, growth had occurred in the Ra culture when the test for nitrite was made. Though all L-phase cultures remained lysogenic, we did not determine whether the inducible phage in L-phase cultures and revertants was the same as that carried by the parent. However, phage from both sources (L-form and parent) lysed a common host (ATCC 19144). P. aeruginosa strains may be multilysogenic and the dominant prophage of the parent could have been lost during induction and/or propagation of L-phage cultures, thereby permitting another prophage to be expressed. The expression of a new prophage could account for the altered antigenic surface of the L-form (12) and possibly even for the melanin-like pigment produced by La. Though Ld remained sensitive to EDTA,

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L-FORMS OF P. AERUGINOSA

189

indicating the presence of residual cell wall, it

Virulence. Though less virulent than the parent culture, the Ld culture caused death in 24 to 48 h of about 50% of the animals when 4 x which in the parent rendered it sensitive to 107 to8 x 107 CFU were injected. The same dose lysozyme. of L. or Lf failed to kill mice; higher doses (2 x

was resistant to the action of lysozyme apparently due to the absence of a mucopeptide layer,

TABLE 4. Antibiotic spectrum of L-forms, revertants, and parent Disk concn (Ag)

Antibiotic

Nalidixic acid

Diam of inhibition zone (mm3) in culturea of: -1 1 1 Ld Lf | CB1 L . j ]CB | +C R. f Rf

R.3

Parent

......

3

11

0

0

0

0

0

0

0

0

Chloramphenicol ...

5 10 30

0 11 21

0 0 13

0 0 0

0 0 0

0 ND 0

0 0 9

0 ND 0

0 ND 0

0 0 10

Tetracycline

........

10 30

ND 27

ND 16

0 15

0 10

ND 23

ND 19

ND 0

0 0

ND 15

Terramycin

........

10 30

24 29

15 20

11 15

9 13

19 23

15 21

9 12

12 17

13 17

DHSM .............

5

13

0

13

16

23

18

27

25

0

Neomycin

5

9

0

15

18

9

0

18

12

0

Gentamicin

10

20

20

22

33

23

20

32

38

17

Kanamycin

5 10 30

0 0 0

0 0 0

0 0 0

13 18 24

0 ND ND

0 ND ND

15 ND ND

0 ND ND

0 0 0

Lincomycin

2

0

0

0

0

0

0

0

0

0

..........

Oleandomycin

......

2

0

0

0

0

0

0

0

0

0

Erythromycin

......

5

0

0

0

0

0

0

0

0

0

Cephaloridine ...... Cephalothin ........ Methicillin ......... Naficillin ........... Cloxacillin ......... Vancomycin ........ Bacitracin .......... Rhondomycin ...... Penicillin .......... Carbenicillin .......

30 30 5

50

0 0 0 0 0 0 0 24 0 0

0 0 0 0 0 0 0 12 0 10

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 17 0 0

0 0 0 0 0 0 0 0 0 11

0 0 0 0 0 0 0 0 0 10

0 0 0 0 0 0 0 0 0 2,000

0 0 0 0 0 0 0 0 0 10

1

1 5 10 5

1oU

Polymyxin

.........

50U

14

13

14

20

26

15

15

13

11

Novobiocin Furadantin

.........

.........

5 50

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

Elkosin ............ Madribon ..........

50 50

10 0

0 0

0 0

0 0

0 0

14 0

0 0

0 0

0 0

Aureomycin ........ Declomycin ........ Viomycin .......... Gantrisin ...........

30 5 2 50 300

27 26 0 10 18

18 16 0 0 0

11 9 0 0 0

11 0 0 0 0

24 24 0 0 ND

17 14 0 0 ND

8 0 0 0 ND

0 0 0 0 ND

13 12 0 0 0

a P4-L broth cultures of Ld and L, tested on P4 agar with and without carbenicillin; L. tested only with carbenicillin in P4 agar; revertants and parent grown and tested in P4 media without carbenicillin. Diameter of disk was 7 mm; all positive results based on duplicate experiments; variation in sensitivities infrequent, never amounting to more than 2 mm. ND, Not done. 'R, grown and tested on TSA.

190

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INFECT. IMMUN.

TABLE 5. Biochemical and other characteristics of L-phase variants, revertants, and parent bacillusa R. Parent Rd Characteristic Lf, Ld Glucoseb ............................

Mannitol ........................... Lactose ............................. Sucrose ............................. Reduction of nitrate to N2 ............ Oxidase ............................

Gelatin liquefaction. Litmus Milk Reduction ........................ Peptonization ..................... Rennet clot ....................... Lysogenicity ........................ Motility ............................ Pyocyanin .......................... EDTA sensitivity .................... Lysozyme sensitivity ................

4+ 4+ alk alk + +



3± 3+ alk alk

+

1+ 1+

alk alk

alk alk

+ +

+ +

1+

4+ 4+ alk alk

alk alk alk

+ +

+ +

1+

2+ 2+ alk alk +

1+

4+

+

+ +

+ +

+b

+ +~

ND ND

ND ND

ND ND

ND ND

ND ND

a Sugar utilizations are recorded for tests in absence of PVP and bovine serum albumin. Exceptions are noted. Cultures were incubated 6 days at 37 C before final determinations were made. Reactions of L-phase were generally slower than for parent or revertant. Symbols: 4, Strong acid; 1, weak acid; alk, alkaline; ND, not done. b Observed in broth cultures; less than 5% motility as tumbling and rotational motion of large cocci, 1 to 1.5

Am. 108 to 4 x 108 CFU) of L. would kill some mice, but an equal dose of Lf failed to produce any apparent signs of illness. With the parent culture, 107 organisms killed approximately 50% of the animals in 24 to 48 h. Both Ld and bacterial forms were isolated from the liver, spleen, kidney, blood, and peritoneal washings of moribund mice. Colony-forming isolates of Ld were about half as numerous as the bacterial forms and occurred only in P4-L agar. Ld isolates reverted quickly on subculture to serotype 3,7,R. The simultaneous isolation of bacteria and L-forms on P4 and P4-L agar, respectively, precludes any conclusion as to whether L-forms existed in vivo. Revertants were observed on stained smears of the peritoneal exudates and blood from dead or moribund mice. The observation that revertants existed predominantly in vivo is in agreement with the fact that L-form isolates on P4-L agar were about 50% fewer than bacterial isolates on P4 agar. This value of 50% is equal to the frequency of reinduction of the revertant to the L-form on P4-L agar stated earlier. When in vivo reversion was studied by administering a single injection of different doses of CB to the mice (1,000 to 50,000 Mg) at the time of inoculation, death from Ld was only delayed and the ratio of L-form to bacterial form in the isolations from the tissues was not altered among the animals receiving different doses of CB. Direct examination of peritoneal washings

by the Dienes technique revealed predominantly L-forms and few bacterial forms in the treated group and only rods in the control group; blood smears of the treated group revealed neither form. With Lf, none of the animals died in either treated or control groups. With LB, CB did prevent death. In the absence of CB, Lb-induced early deaths yielded confluent L-form growth on cultures of peritoneal washings on P4-L agar and few bacteria isolated on P4 agar. The longer death was delayed the greater was the number of revertants isolated with the L8 forms. These revertants serotyped as 3,6,; the in vitro cultivated revertants (Table 3) typed 1,6,7,R,J,L; the original parent cells were type 3. When mice infected by Ld (2 x 108 CFU) were given repeated injections of CB (six injections at intervals of 12 h), deaths were prevented. All mice not receiving CB (control group) died within 24 to 36 h. From one mouse, dead 4 days after termination of CB therapy, revertants were isolated. From the remaining mice, sacrificed 2 weeks after inoculation, only the L-form was recovered on CB-containing media. Passage of Ld in mice given 15,000 Mg of CB at time of infection to prevent death yielded a stable Ld (3,7,R) but were completely CB dependent for growth. This derivative was isolated from the spleen 2 weeks after inoculation. The dependent form of Ld killed mice at a dose of 2 x 108 CFU. No revertants were isolated from dead or moribund mice; only L-forms grew out on P4-L agar.

VOL. 11, 1975

L-FORMS OF P. AERUGINOSA

In the absence of bacterial isolates, the isolation of L-forms on the inductive medium (P4-L) would not suggest reinduction to the L-form of bacterial revertants present in the organs. In mice surviving lower doses of the CB-dependent isolate, the L-form continued to be isolated from mice for up to 2 weeks post-inoculation in the absence of bacterial isolations. An attenuated, CB-independent derivative (also 3,7,R serotype) was subcultured from the dependent form. The attenuated form (nonlethal for mice) survived at least 2 weeks in the peritoneal cavity and retained its serotype.

DISCUSSION Three L-forms (Ld, b8, and Lf) of a virulent strain of P. aeruginosa could be distinguished from one another by bacteriological properties, mouse virulence, and type-specific antisera prepared against somatic antigens of P. aeruginosa strains. Though Lb failed to agglutinate in the antisera tested, it retained a capacity to synthesize cell wall precursors retaining some surface antigen of the parent (serotype 3,7) as demonstrated by the serotype of its revertant (serotype 1,6,7). Unlike Lb, the revertants of Ld and Lf carry the same antigens as their respective L-forms. Weibull et al. (22), investigating the properties of the three L-forms of Proteus mirabilis strains and their respective parents, found no serological relationship between one of the L-forms and its parent bacterium. Weibull et al. (22) report that Dienes et al. worked with another L-form of the same Proteus strain that was not agglutinated by antiserum against the parent bacterium nor was the bacterium agglutinated by antiserum against the derived L-form. Our results suggest that from a single bacterial strain different L-forms may be induced that may or may not be serologically related to the parent. These serological changes in the L-form cultures warn against assuming homogeneity of L-forms derived from a common ancestral parent. To determine whether "antigenic modulation" of L-forms and revertants occurs at time of induction, the reisolation of the three L-forms was attempted. Preliminary study on the reisolation via sucrose medium (gradient technique) yielded an L-form as slowly growing as the original Lb. However, the early transfers were serotype 3,7,R and more virulent than the strain tested after prolonged passage. Whereas the modulation reported for Lb and b, remained constant for later passages, evidence is not at hand on their early characteristics. Thus far, the serotype of the Ld culture from passages 6 to

191

30 has remained the same. Lb first typed at passage 32, though, was already changed antigenically to 1,4,6,R,J,L. Our results suggest that variation in serotype could conceivably result from in vivo production of wall-weakened bacterial forms or Lphase persisters, surviving long enough to allow reversion to a new serotype. Recurrent infections have been reported to occur with organisms of the same or different serological strain (20) and have normally been attributed to exogenous infection. However, antigenic modulation occurring in vivo via "persister" phase would have survival advantages for the invader, for a primary immune response mounted to the antigenic determinants of a primary invader would not be as effective against the emergent variant. In a hospital study (13), it was reported that over a period of 15 months, recovery of type 7 varied inversely with recovery of type 3, which shared minor antigens with type 7. Changes in serotype of some strains of P. aeruginosa have occurred in vitro during long preservation on agar at room temperature (8). L-forms can be markedly different in their antibiotic sensitivities from each other as well as from their common parent, and revertants can acquire new antibiotic resistances in the absence of previous exposure to the drug (10). L8 failed to agglutinate in typing sera and is the only L-form showing greatly enhanced sensitivity to polymyxin. This suggests a virtual absence of outer cell wall components, which would facilitate greater access by polymyxin to the membrane, the proposed site of its action (15). Alternatively, polymyxin is reported to have an affinity for the phospholipids of cell membranes (18), so that enhanced sensitivity of b, may result from changes in lipid constitution of its membrane (1, 2). This may explain why the stable L-form of P. aeruginosa induced by Hubert et al. (9) was more resistant to polymyxin B than the parent. The common finding that L-forms are generally more sensitive than the parent to many of the antibiotics that have their primary site of action within the protoplasmic body cannot always be attributed to greater permeability, for R8, with its intact cell wall, showed greater sensitivity to gentamicin than its nonagglutinating, cell wall-deficient L-form. Depending on the antibiotic, then, changes in sensitivity may result not only through removal of cell wall but also through such physiological and structural alterations affecting permeability as changes in lipid content, in strength of crosslinkages in cell wall or fluidity of lipid layers, or in changes in the hydrophobic-hydrophilic char-

192

BERTOLANI, ELBEFR,tG, AND RALSTON

INFECT. IMMUN.

by the Bureau of Medicine and Surgery, U.S. Navy, and by acter of the lipid envelope. The three L-forms were less virulent than the the Naval Biomedical Research Laboratory. parent and showed different virulences among LITERATURE CITED themselves. Slow growth rate and lack of outer 1. Brown, M. R. W., and J. Melling. 1969. Role of divalent cell wall components, characteristics of L., did cations in the actions of polymyxin B and EDTA on Pseudomonas aeruginosa. J. Gen. Microbiol. not necessarily result in loss of virulence for 59:263-274. mice, for Lb was slightly virulent whereas Lf was 2. Brown, M. R. W., and W. Watkins. 1970. Low magnesium not. Osmotic lability does not seem to be a and phospholipid content of cell walls of Pseudomonas factor in loss of virulence, since Ld, the most aeruginosa resistant to polymyxin. Nature (London) 227:1360-1361. osmotically sensitive of the three L-forms, was 3. Carter, W., and K. S. McCarty. 1966. Molecular mechalso the most virulent. anisms of antibiotic action. Ann. Intern. Med. Few rodlike forms were seen on direct exami64:1089-1113. nation of peritoneal fluid and blood taken at 4. Clasener, H. 1972. Pathogenicity of the L-phase of bacteria. Annu. Rev. Microbiol. 26:55-84. death of mice receiving CB simultaneously with manual. 1953. 9th ed. Difco Laboratories Inc., Ld forms. This suggested that reversion may 5. Difco Detroit. indeed not be a requirement for death after Ld 6. Fisher, M. W., H. B. Devlin, and F. J. Gnabasik. 1969. infection. Supporting this was the isolation New immunotype schema of Pseudomonas aeruginosa based on protective antigens. J. Bacteriol. 98:835-836. from mice treated with CB of nonreverting, G. W., and S. G. Wilkinson. 1965. The effect of CB-dependent, virulent derivatives of Ld on 7. Gray, ethylenediaminetetraacetic acid on the cell walls of P4-L agar in the absence of bacterial isolates on some gram-negative bacteria. J. Gen. Microbiol. the noninductive P4 agar. These two pieces of 39:385-399. experimental evidence favor the idea that death 8. Homma, J. Y., H. Shionoya, H. Yamada, M. Enomota, and K. Miyao. 1972. Changes in serotype of Pseudomomay occur in the absence of reversion of Ld in nas aeruginosa. Jap. J. Exp. Med. 42:171-172. vivo. Opposed to these observations was the 9. Hubert, E. C., C. S. Potter, T. J. Hersley, M. Cohen, G. finding that administration of multiple doses of M. Kalmanson, and L. B. Guze. 1971. L-forms of Pseudomonas aeruginosa. Infect. Immunity 4:60-72. CB during an Ld infection prevented death of Z. N., M. M. Dykhno, N. G. Prozanovsky, the mice. Thus our evidence for the former 10. Kochemasova, and D. Ya Bakanova. 1969. L-transN. Kassirskaya, G. hypothesis may not be definitive because of a formation of mycobacteria tuberculosis as one of the quantitative aspect. sources of development of drug resistant strains. Vestnik. Akademii Med. Nauk SSSR 24:152-159. It may be that the virulent, CB-dependent J. W., and J. T. Douglas. 1973. Induction and derivative of Ld is present in the original Ld 11. Lawson, reversion of the L-form of Neisseria gonorrhoeae. Can. culture at sublethal doses. Thus there may be J. Microbiol. 19:1145-1151. two factors important in causing death. (i) 12. Liu, P. V. 1969. Changes in somatic antigens of Pseudomonas aeruginosa induced by bacterio-phages. J. InReversion may account for deaths from the Ld fect. Dis. 119:237-246. culture and may be prevented by multiple doses 13. Moody, M. R., V. M. Young, D. M. Kenton, and G. D. of CB; and (ii) death may occur in the absence Vermeulen. 1972. Pseudomonas aeruginosa in a center of reversion due to as yet unknown virulence for cancer research. I. Distribution of intra species types from human and environmental sources. J. Infect. Dis. factors associated with nonreverting L-forms if 125:95-101. inoculated in sufficiently large numbers. We Z. 1973. Enzymatic and toxigenic activity of have earlier observed that a normally lethal 14. Muszynski, culture filtrates of high and low virulent strains of dose of Ld in the living state, when heat Pseudomonas on mice. Pathol. Microbiol. 39:135-147. inactivated or lysed, loses its lethality, suggest- 15. Newton, B. A. 1958. Surface active bactericides, p 62-92. In 8th Symp. Soc. Gen. Microbiol. Cambridge Univering viability as a determinant of virulence of Ld sity Press, Cambridge. units (cf. 14). 16. Repaske, R. 1956. Lysis of gram negative bacteria by The isolation of nonreverting (CB-dependent) lysozyme. Biochim. Biophys. Acta 22:189-191. L-forms of Ld 2 weeks post-inoculation from 17. Ried, L. J., and H. Muerch. 1938. A simple method of estimating fifty percent end points. Amer. J. Hyg. mice receiving CB at the time of injection of a 27:493-497. that the suggests of the Ld culture lethal dose 18. Salton, M. R. 1961. The anatomy of the bacterial surface. L-form may potentiate a chronic infection. In Bacteriol. Rev. 25:77-99. one such mouse it was found that despite the 19. Symposium on carbenicillin: a clinical profile. 1970. J. Infect. Dis. 122(Suppl.):S1-S116. lack of any apparent clinical signs of illness, 2 on oral indanyl carbenicillin in the treatment weeks after inoculation of Ld one kidney yielded 20. Symposium of urinary tract infections. 1973. J. Infect. Dis. revertant growth, possibly via in vivo develop127(Suppl.) :S93-S165. ment of CB-independent, attenuated forms as 21. Turck, M., K. N. Anderson, and R. G. Petersdorf. 1966. Relapse and infection in chronic bacteriuria. N. Engl. described earlier in this report. J. Med. 275:70.

ACKNOWLEDGMENTS This investigation was supported by funds made available

22. Weibull, C., W. D. Bickel, W. T. Haskins, K. C. Milner, and E. Ribi. 1967. Chemical, biological, and structural properties of stable Proteus L forms and their parent bacteria. J. Bacteriol. 93:1143-1159.

Variations in properties of L-forms of Pseudomonas aeruginosa.

INFEcTiON AND IMMUNITY, Jan. 1975, p. 180-192 Copyright i 1975 American Society for Microbiology Vol. 11, No. 1 Printed in U.S.A. Variations in Prop...
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