Vol. 25, No. 11JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1987, p. 2154-21580095-1137/87/112154-05$02.00/0Copyright C 1987, American Society for Microbiology

Spheroplast Induction in Clinical Isolates of Serratia marcescens inthe Presence of Ca2+ or Mg2+YOSHIFUMI TADA* AND JUNJI YAMAGUCHI

Department of Bacteriology, Kinki University School of Medicine, Sayama-cho, Minamikawachi-gun, Osaka 589, Japan

Received 27 May 1987/Accepted 5 August 1987

Serratia marcescens was easily induced to form spheroplasts by P-lactam antibiotics in the presence of Ca2+or Mg2+ without an osmotic stabilizer such as sucrose. The spheroplasts grew in volume, although they couldnot divide. They were stable for more than 10 h at 37°C in a medium containing a high concentration ofantibiotic, and they had the ability to revert to the original bacillary form. Ca2+ was more effective inspheroplast induction than Mg2+. The effect was proportional to the concentration of cations. In 40% of 180clinical isolates of S. marcescens, more than 40% of the original bacterial cells were induced to formspheroplasts by ceftizoxime in a medium supplemented with 40 mM Ca2+. A high spheroplast induction ratewas observed even in medium with 10 mM Ca2+. Few isolates that were supersusceptible to ceftizoxime (MIC,<0.2 ,ig/ml) were induced to form spheroplasts at a high rate. No difference in spheroplast induction rate orextent between antibiotic-resistant strains and relatively susceptible strains (MIC, >0.2 ,ig/ml) was found. Theserotype of S. marcescens had no effect on the spheroplast induction rate. Monocations (Na' and K+) had littleeffect on spheroplast induction.

During the past decade, Serratia marcescens and someother gram-negative bacteria have been recognized as im-portant opportunistic or nosocomial pathogens, particularlyof the urinary and respiratory tracts (2, 13, 17). Chemother-apy with antibiotics often results in failure, especially incompromised hosts. The lack of clinical response wasmainly due to the development of resistance in pathogenicbacteria (5, 16, 20). However, since L forms and sphero-plasts induced by P-lactam antibiotics were not eliminatedby the drugs because of loss of the cell wall, there is a fairchance for reinfection by them (3, 6, 7, 10).

Generally, cell wall-deficient bacteria such as spheroplastsare induced under high osmotic pressure. It has thereforebeen supposed that they were not easily induced in vivobecause of isotonic conditions. Previous to 1970, a fewauthors had reported that osmotic lysis of cell wall-deficientbacteria induced in hypertonic medium was prevented bylow concentrations of cations (4, 14, 15, 18). In the presentpaper, we report that high osmotic pressure is unnecessaryfor the induction of stable spheroplasts in clinical isolatesdue to the presence of Ca2+ or Mg2+. We also report acorrelation between spheroplast induction and the drugsusceptibility or serotype of S. marcescens.

MATERIALS AND METHODS

Bacteria. The bacterial strains used in this study wereclinical isolates of S. marcescens, except for S. marcescensIAM1184. Strains of the KUS series were isolated in theDepartment of Central Clinical Laboratory, Kinki Univer-sity Hospital. Strains of the TES series, isolated in fivedifferent hospitals, were obtained from S. Kimura, TeikyoUniversity, Tokyo, Japan. S. marcescens KUF was derivedfrom the pus of a surgical patient and cultured in ourlaboratory for several years. S. marcescens IAM1184 was a

* Corresponding author.

laboratory strain originating from the culture collection ofthe Institute of Applied Microbiology, University of Tokyo.Media and culture. Brain-heart infusion (BHI) broth

(BHIB; Nissui Pharmaceutical Co., Tokyo) was used as thebasal medium. The basal medium was supplemented with aconcentrated solution of CaCl2 or MgSO4 to give appropriatefinal concentrations. They were designated induction me-dium, BHIB-Ca, or BHIB-Mg and prepared just before theexperiments. All cultures were incubated aerobically at 37°Cthroughout this study. BHI agar (0.8% agar) was used fordetermination of CFU for spheroplasts.

Antibiotics. Ceftizoxime (CZX) was a gift of FujisawaPharmaceutical Co., Osaka, and cefotetan (CTT) was a giftof Yamanouchi Pharmaceutical Co., Tokyo. Carbenicillin(CBPC) was purchased from Sigma Chemical Co. All anti-biotics were solubilized in distilled water just before theexperiments.MIC determinations. All strains were tested for suscepti-

bility to antibiotics by the agar dilution technique on BHIagar. About 106 bacteria were spotted on plates containingserial twofold dilutions of antibiotics. The plates were incu-bated at 37°C overnight. The MIC was defined as the lowestconcentration of antibiotic that inhibited visible growth ofthe bacteria.

Spheroplast induction. Organisms were cultured over-night. They were transferred to fresh BHIB and cultured for3 to 4 h. The exponentially growing cells were incubated inmedium with P-lactam antibiotics. The final inoculum sizewas approximately 107 CFU/ml. The concentration of anti-biotic was 10 times higher than the MIC.CFU of spheroplasts. Spheroplasts were gently diluted

with BHIB-Ca not containing antibiotics. Diluted cell sus-pension was plated on agar medium supplemented with 20mM CaCl, (reversion medium) and incubated for 24 h.Spheroplasts reverted to bacillary forms, and colonies onagar were counted (19).

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SPHEROPLAST INDUCTION IN S. MARCESCENS 2155

FIG. 1. Spheroplasts induced in BHIB supplemented with Mg2+by CZX. Newborn spheroplasts are small, and the surface issmooth. Bar, 1.0,m.

Celi number of spheroplasts. The number of spheroplastswas directly counted by using a hemacytometer (ImprovedNeubauer).

Although S. marcescens is small to be counted under themicroscope, in several hours viable spheroplasts grewenough to be counted by hemacytometer. Cultures wereadded to the same volume of trypan blue solution (1%). After10 to 30 min, unstained cells were counted.

Serotyping. The serotype of strains was determined by anagglutination test (11), with the specific antisera to O anti-gens of S. marcescens (Denka Seiken Co., Ltd., Tokyo).Overnight cultures of each strain on BHI agar were sus-pended in physiological saline and killed by heating at 120°Cfor 20 min. After cooling, the bacterial suspensions werecentrifuged at 3,000 rpm, and the sediments were suspendedin physiological saline at a concentration of about 1 mg/mlfor use as antigens. Samples (0.5 ml) of the antigens werepoured into small test tubes containing 0.1 ml of specificantisera, and the test tubes were placed overnight in arefrigerator after incubation at 50°C for 2 h. The antigen thataggregated macroscopically in one test tube was designatedthe O antigen corresponding to the specific O antiserum inthe test tube.

Scanning electron microscopy. An exponentially growingculture of S. marcescens KUF was incubated in BHIBcontaining CZX and 40 mM Mg2 for 4 h. The spheroplastsobtained were washed twice with 0.1 M phosphate buffer(pH. 7.2) supplemented with 20 mM Mg2+. Cells wereprefixed with 4% glutaraldehyde in the same buffer for 1 h at4°C. After being washed with the same buffer, cells werepostfixed with 1% OS04 for 1 h at 4°C. The fixed cells were

dehydrated in a graded series of ethanol. They were dried bythe critical-point drying method with liquid carbon dioxide.Then the cells were spattered with a thin (ca. 20-nm) layer ofgold. Preparations were examined in an electron microscope(JSM 840) operating at 10 kV.

Osmotic pressure. The osmotic pressure was determinedby measurement of the freezing point of depression (AUTO& STAT OM-6010; Kyoto Daiichi Kagaku Co., Kyoto,Japan).

RESULTS

Spheroplast induction in BHIB-Ca and BHIB-Mg. Sphero-plast induction occurred within 2 h. There was no sphero-plast increase in liquid medium, but cell volume increasedduring further incubation with the ,-lactam antibiotic (Fig.1). Spheroplast induction rates in six strains of S. marces-cens were proportional to the concentration of Ca2+ or Mg2+(Table 1). The induction rate was represented by the per-centage of original bacillary-form cells that were induced toform spheroplasts. High induction rates (over 50%) werefound in an induction medium (BHIB) supplemented with 40mM Ca2+ (BHIB-Ca [40 mM]). Strains of S. marcescenswere not induced to form spheroplasts in a medium notsupplemented with Ca2+ or Mg2+ except S. marcescensKUF. Comparing the effect of Ca2+ and Mg2+ on sphero-plast induction, Ca2+ was more effective than Mg2+. Somestrains (S. marcescens KUF, KUS001, and KUS003) wereinduced to form spheroplasts at relatively high rates even ina medium with 10 mM Ca2+, but others (strains KUS002 andKUS004) were hardly induced to form spheroplasts underthe same conditions.

Stability of spheroplasts induced in BHIB-Ca. Exponen-tially growing cells of six strains were incubated in BHIB-Ca(40 mM) containing CZX. The concentration of CZX was 10times higher than the MIC. Variations in cell number andCFU of spheroplasts during incubation in induction mediumare shown in Fig. 2. In three strains, S. marcescens KUF,KUS001, and KUS003, there was only a slight decrease inthe cell number of spheroplasts for at least 10 h in a mediumsupplemented with Ca2" and CZX. In other strains, arelatively marked decrease in cell number was found, but aconsiderable number, more than 105 cells per ml, of sphero-plasts remained even after incubation for 10 h. The pattern ofvariation in spheroplast CFU was essentially similar to thatof the cell number mentioned above. When the bacillichanged to spheroplasts in BHIB-Ca, the cell number andCFU quickly decreased, but after further incubation only aslight decrease was shown. It must be noted that about 10%of the original cells maintained their viability even after 10 hof incubation.

TABLE 1. Spheroplast induction rates' in BHIB supplementedwith Ca2 + or Mg2 +

S. marcescens Induction rate (%) at cation concn:Cationstrain 40 mM 20 mM 10 mM 0

KUF Ca2+ 66 34 22 10Mg2+ 40 28 15

IAM1184 Ca2+ 41 28 15 0Mg2+ 19 13 4

KUS001 Ca2+ 65 40 28 0Mg2+ 43 33 23

KUS002 Ca2+ 25 7 2 0Mg2+ 12 8 2

KUS003 Ca2+ 63 41 26 0Mg2+ 36 27 17

KUS004 Ca2+ 22 14 1 0Mg2+ 14 7 1

'Percentage of original bacillary cells forming spheroplasts. Cell numberwas counted by using a hemacytometer. Spheroplasts were induced by CZX.

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INCUBATION TIME (Hours)

FIG. 2. Variation of cell number and CFU of spheroplasts in

BHIB supplemented with Ca2+. At time zero, CZX was added.Spheroplast induction was complete after 4 h of incubation. Sym-bols: O, KUF; *, IAM1184; O, KUSOO1; M, KUSOO2; A, KUSOO3;A, KUSOO4.

Effects of monovalent cations on spheroplast induction.Monovalent cations (Na' and K+) had little effect on sphero-plast induction (Table 2). The induction rate in BHIB-Ca (10mM) was from 21 to 26% of that in three strains that were

easily induced to form spheroplasts, but a low induction ratewas found in BHIB-Na (40 mM) and BHIB-K (40 mM). Theosmotic pressure in BHIB-Na (40 mM) and BHIB-K (40mM) was 387 and 385 mosM/liter, respectively, and was

higher than that in BHIB-Ca (10 mM). However, the induc-tion rate in BHIB-Na (40 mM) and BHIB-K (40 mM) was not

TABLE 2. Effect of Ca2+, Na', and K+ added to BHIBon spheroplast induction

Cation and Osmotic Spheroplast induction rate (%)concn

pressureconcn (mosM/ S. marcescens S. marcescens S. marcescens

(mM) liter) KUF KUSOO1 KUS003

Ca2+ (40) 393 67 60 53Ca2+ (10) 347 21 26 22Na' (40) 387 8 4 2K+ (40) 385 8 6 4None 330 5 1 1

TABLE 3. Spheroplast induction by CBPC, CZX, and CTT

Induction rate (%)S. marcescens strain

CBPC CZX CTT

KUF 67 56 44IAM1184 47 19 16KUS001 NDa 65 38KUS002 ND 12 12KUS003 ND 64 50KUS004 ND 9 13

a Strains were so resistant to CBPC that spheroplast induction was notdetectable (ND).

higher than that in BHIB-Ca (10 mM), showing that theeffect of divalent cations on spheroplast induction was notdue to a rise in osmotic pressure.

Spheroplast induction rates by several P-lactam antibioticsin BHIB-Ca. As was the case with CZX, S. marcescens wasinduced to form spheroplasts at high rates in BHIB-Ca (40mM) by CBPC or CTT, although induction rates betweenantibiotics varied (Table 3).

Spheroplast induction rates in clinical isolates by CZX. Atotal of 180 clinical isolates of S. marcescens were subjectedto spheroplast induction in BHIB-Ca (40 mM) and BHIB-Ca(10 mM) in the presence of CZX (Table 4). Forty (22%) of180 isolates were induced to form spheroplasts at a high rate(over 40%) when 40 mM Ca2+ was added to the inductionmedium, and 83 isolates, about half of all isolates, wereinduced at a rate of over 20%.On the other hand, despite considerable reduction in the

induction rate in BHIB medium supplemented with 10 mMCa2+, 11 isolates (6%) were induced at a high rate, and aninduction rate of over 20% was found for 31 isolates (17%).

Spheroplast induction rate and antibiotic susceptibility inclinical isolates. The MICs for all isolates were determined bythe method mentioned in the text. According to their sus-ceptibility to CZX, isolates were divided into four groups:superresistant (group 1), resistant (group 2), susceptible(group 3), and supersusceptible (group 4). Spheroplast in-duction rates of the isolates in the four groups were exam-ined (Table 5). An apparent difference in the induction ratesamong the four groups was found between the supersuscept-ible isolates and the others. Few supersusceptible isolateswere induced to form spheroplasts at rates of over 40%,although over 24% of the isolates in the other groups wereinduced at this rate. Only 1/10 of the supersusceptibleisolates were induced to form spheroplasts at a rate of over20%, but half of the isolates in the other groups were inducedat this rate. These results indicated that supersusceptibleisolates were hardly induced to spheroplasts, but in suscep-tible, resistant, and superresistant isolates, the difference inantibiotic susceptibility had no correlation with the sphero-plast induction rate.

TABLE 4. Spheroplast induction rates of clinical isolates ofS. marcescens in the presence of Ca2+

No. (f) of strainsInduction rate ff)

40 mM CaC12 10 mM CaCI2

>40 40 (22) 11 (6)40-30 16 (9) 7 (4)29-20 27 (15) 13 (7)19-10 32 (18) 32 (18)

<10 65 (36) 117 (65)

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SPHEROPLAST INDUCTION IN S. MARCESCENS 2157

TABLE 5. Spheroplast induction rate and antibiotic susceptibility

No. (%) of isolates with spheroplastGroupa MIC induction rate:(~Lg/mI)

>40% 40-20Wo <20%

1 >100 20 (28) 24 (34) 27 (38)2 50-12 4 (24) 3 (18) 10 (58)3 6-0.2 15 (28) 14 (26) 24 (45)4 <0.2 1 (2) 3 (8) 35 (90)a Isolates were divided into four groups based on susceptibility to CZX.

Serotype and spheroplast induction of clinical isolates. Atotal of 30 clinical isolates were serotyped by O agglutination(Table 6). Half of the isolates were induced to form sphero-plasts at a high rate (over 50%, group A), and the others werehardly induced (group B). Seven of 30 isolates were nontyp-able strains. Serotypes 01, 03, and 017 were found only ingroup A, but 04, 06, 012/13, and 013 were found in bothgroups. The specific and common serotype in the isolateswhich were induced to form spheroplasts at a high rate wasnot found. These results indicated no correlation betweenserotype and spheroplast induction.

DISCUSSIONS. marcescens was induced to form stable spheroplasts by

,-lactam antibiotics in the presence of Ca2, or Mg2+. Theprocess of morphological change in spheroplast inductionwas similar to that in a hypertonic medium (1). The concen-tration of Ca2+ and Mg2+ was relatively high but had littleeffect on variations in the osmotic pressure in the medium.Unlike divalent cations, Na' or K+ at the same concentra-tion had less effect on spheroplast induction (Table 2). Thesefacts indicate that the effects of Ca2" or Mg2+ do not dependon enhanced osmotic pressure. Ca2+ was about twice as

effective as Mg2" in spheroplast induction. Spheroplastscould revert to their original bacillary forms. These facts alsoshowed that the effect of Ca2" or Mg2+ did not result fromsome physical reaction.Compared with the number of spheroplasts induced by

CZX at 10 times the MIC, the number ofCFU was very low.All viable spheroplasts, however, do not always revert inthis reversion medium. It therefore remains a possibility thatmore spheroplasts revert on the reversion medium if it isimproved (8, 9).Many isolates from several hospitals in Tokyo and Osaka

were induced to form spheroplasts at a high rate in mediumincluding from 10 to 40 mM Ca2+. No significant differencewas seen in the rate and extent of induction among theisolates from different regions. S. marcescens also may beinduced to form spheroplasts in vivo in sites of relativelyhigh Ca2+ and Mg2+ concentrations, like the urinary tract,when treatment with P-lactam antibiotics is given.The concentration of Ca2+ and Mg2+ in the urine of normal

humans is at most 10 mM collectively, so 10 to 40 mM Ca2+and Mg2+ seems to be a considerably high concentration.However, the concentration of Ca2+ and Mg2+ in the urinarytract is increased when urine flow is slowed by urinarycalculi or insertion of urethral catheters.Although spheroplast induction was significantly de-

creased in isolates that were supersusceptible to CZX,resistant isolates were induced at a high rate regardless ofthe grade of susceptibility to CZX. This may make the cureof infectious diseases caused by S. marcescens difficult.Many excellent 1-lactam antibiotics have been developed.

However, they are not always effective in disease, especiallyin a complicated urinary tract infection, despite their con-siderable antimicrobial activity in vitro (12). Opportunisticinfection after treatment with P-lactam antibiotics also oc-curred frequently (21). Although the emergence of multidrug

TABLE 6. Serotype and spheroplaSt induction in clinical isolates

S. marcescens Spheroplast induction in serotype:strain 01 02 03 04 05 06 07 08 09 010 011 012 013 014 015 016 017

Group ATESO10 + +TESO18 +TESO21 +TESO36 +TESO46 +TES125 +TES144 +KUSOO1 + +KUSOO3 +KUSO11 +KUSO19 +KUSO20 + +

Group BTESOO6 + +TESOO7 +TESO11 +TESO13 +TESO47 +TESOSO +TES106 +TES113 +TES134 +TES138 + +TES139 +

a Group A, Isolates that were induced to form spheroplasts at high rate; group B. isolates that were hardly induced to form spheroplasts.

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2158 TADA AND YAMAGUCHI

resistance during chemotherapy is an increasingly recog-nized cause of antibiotic failure, spheroplast induction invivo during chemotherapy should also be a reason forantibiotic failure. S. marcesc ens was induced to formspheroplasts at the sites where relatively high concentrationsof Ca2" and Mg2" were accumulated, and the spheroplastswere no longer susceptible to P-lactam antibiotics because ofthe loss of the cell wall. The presence of high concentrationsof Ca2" and Mg2" permits persistence of spheroplasts, andthey may revert to the original bacillary forms after chemo-therapy has been stopped. Cell wall-deficient bacteriumlikespheroplasts have long been suspected of causing pathogen-esis.We examined several strains of other gram-negative bac-

teria. Spheroplast induction by CZX and Ca2+ was alsofourild in Escherichia coli, Salmonella enteritidis, and Pro-teus mirabilis. This phenomenon may explain some cases ofintractable and opportunistic infection after chemotherapy.

LITERATURE CITED1. Furutani, A., Y. Tada, and J. Yamaguchi. 1985. Reversion to

bacillary forms of Serratia marcescens spheroplasts induced bytarbenicillin. Microbiol. Immunol. 29:901-907.

2. Geiseler, P. J., B. Harris, and B. R. Anderson. 1982. Nosocomialoutbreak of nitrate-negative Serratia marcescens infections. J.Clin. Microbiol. 15:728-730.

3. Guze, L. 1. 1964. Persistence of bacteria in protoplast form afterapparent cure of pyelonephritis in rats. Science 143:1340-1341.

4. Harold, F. M. 1964. Stabilization of Streptococcus faecalisprotoplasts by spermine. J. Bacteriol. 88:1416-1420.

5. John, J. F., and E. F. McNeill. 1981. Characteristics of Serratiamarcescens containing a plasmid coding for gentamicin resis-tance in nosocomial infections. J. Infect. Dis. 143:810-817.

6. Kalmanson, G. M., and L. B. Guze. 1968. Pyelonephritis;isolation of protoplasts from human kidney tissues, p. 406-414.In L. B. Guze (ed.), Microbial protoplasts, spheroplasts, andL-forms. The Williams & Wilkins Co., Baltimore.

7. Kaye, D. 1975. Host defense mechanisms in the urinary tract.Urol. Clin. N. Am. 2:407-422.

8. Landman, O. E., and A. Forman. 1969. Gelatin-induced rever-

sion of protoplasts of Bacillus subtilis to the bacillary form. J.Bacteriol. 99:576-589.

9. Landman, O. E., A. Ryter, and C. Frehel. 1968. Gelatin-inducedreversion of protoplasts of Bacillus subtilis to the bacillary form:electron-microscopic and physical study. J. Bacteriol. 96:2154-2170.

10. Mitchell, P. D., and K. Parent. 1982. Role of cell wall deficientbacteria in diseases, p. 383-409. In G. J. Domingue (ed.), Cellwall deficient bacteria. Addison-Wesley Publishing Co., Inc.,Reading, Mass.

11. Okuda, T., N. Endo, Y. Osada, and H. Zen-yoji. 1984. Outbreakof nosocomial urinary tract infections caused by Serratia mar-cescens. J. Clin. Microbiol. 20:691-695.

12. Penn, R. G., L. C. Preheim, C. C. Sanders, and D. K. Giger.1983. Comparison of moxalactam and gentamicin in the treat-ment of complicated urinary tract infections. Antimicrob.Agents Chemother. 24:494-499.

13. Platt, D. J., and J. S. Sommerville. 1981. Serratia speciesisolated from patients in a general hospital. J. Hosp. Infect. 2:341-348.

14. Razin, S. 1964. Factors influencing osmotic fragility of Myco-plasma. J. Gen. Microbiol. 36:451-459.

15. Rodwell, A. W. 1965. The stability of Mycoplasma mycoides. J.Gen. Microbiol. 40:227-234.

16. Sanders, C. C. 1986. Emergence of resistance to lP-lactams,aminoglycosides, and quinolones during combination therapyfor infection due to Serratia marcescens. J. Infect. Dis. 153:617-619.

17. Sifuentes-Osornio, J., G. M. Ruiz-Palacios, and D. H. M.Groschel. 1986. Analysis of epidemiologic markers of noso-comial Serratia inarcescens isolates with special reference tothe Grimont biotyping system. J. Clin. Microbiol. 23:230-234.

18. Tabor, C. W. 1961. Stabilization of protoplasts and spheroplastsby spermine and other polyamines. J. Bacteriol. 83:1101-1111.

19. Tada, Y., and J. Yamaguchi. 1983. Necessity for de novo DNAsynthesis for reversion of Escherichia coli spheroplasts tobacillary forms. Microbiol. Immunol. 27:893-896.

20. Tompkins, L. S., J. J. Plore, and S. Falkow. 1980. Molecularanalysis of R-factors from multiresistant nosocomial isolates. J.Infect. Dis. 141:625-636.

21. Yu, V. L. 1981. Enterococcal superinfection and colonizationafter therapy with moxalactam, a new broad-spectrum antibi-otic. Ann. Intern. Med. 94:784-785.

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