ANTIMICROBLAL AGENTS AND CHEMOTHERAPY, Nov. 1992, p. 2375-2380
0066-4804/92/112375-06$02.00/0 Copyright © 1992, American Society for Microbiology
Vol. 36, No. 11
Comparison of the Antibacterial Efficacies of Ampicillin and Ciprofloxacin against Experimental Infections with Listeria monocytogenes in Hydrocortisone-Treated Mice MARC L. vAN OGTROP,1'2 HERMAN MATTIE,1 BEATA RAZAB SEKH,1 ELISABETH vAN STRIJEN,1 AND RALPH VAN FURTH'* Department of Infectious Diseases, University Hospital, 1 and J. A. Cohen Institute for Radiopathology and Radiation Protection, 2 Leiden, The Netherlands Received 3 February 1992/Accepted 12 August 1992
The efficacies of ciprofloxacin and ampicillin against Listeria monocytogenes in an immunosuppressed mouse model of listeriosis were compared. Immunosuppression was achieved by administration of 2.5 mg of hydrocortisone acetate daily. Both ciprofloxacin and ampicillin were effective in reducing the number of viable L. monocytogenes cells in the liver and spleen. After treatment with 100 mg of ampicillin per kg of body weight every 6 h for 3 days, virtually no L. monocytogenes could be recovered from the livers and spleens of the mice. In contrast, after treatment with 100 mg of ciprofloxacin per kg every 6 h for 3 days, a geometric mean of 5 x 10 CFU of L. monocytogenes was recovered from the spleens and 1 x 10i CFU was recovered from the livers of the mice. Results of the study show that the antibacterial efficacy of ampicillin is far superior to that of ciprofloxacin in our animal model of listeriosis.
Listeria monocytogenes is a facultative intracellular bacterial pathogen that causes severe infections, such as septicemia and meningitis, which can be life threatening in immunocompromised patients. The patients most often affected by severe listeriosis are neonates, transplant recipients, patients on glucocorticosteroids, and patients with lymphomas (1). Standard therapy for listeriosis consists of either ampicillin or penicillin, sometimes combined with an aminoglycoside (1); there are few well-documented alternatives for the treatment of listeriosis. Co-trimoxazole, vancomycin, tetracycline, chloramphenicol, and cephalothin have all been suggested as alternatives to ampicillin (1), but controlled studies have not been performed. Ciprofloxacin is a fluorinated quinolone with in vitro bactericidal activity against L. monocytogenes (4); moreover, it easily penetrates phagocytic cells (5, 7, 8). These characteristics make ciprofloxacin an interesting alternative therapy for listeriosis. In the present study, the antimicrobial efficacy of ciprofloxacin was compared with that of ampicillin for the treatment of an experimental infection with L. monocytogenes in mice immunocompromised by treatment with glucocorticosteroids. The design of the study was, first, to determine the dosage interval for the treatment of this experimental infection by using a maximally effective dose of ampicillin or ciprofloxacin, then to determine the maximally effective dose of either drug after a single dose, and finally, to determine the antibacterial efficacy of both antimicrobial drugs by giving the maximally effective doses at the determined dosage intervals for a period of 3 days. (This study was presented in part at the 5th European Congress of Clinical Microbiology and Infectious Diseases, Oslo, Norway, 1991.)
MATERIALS AND METHODS Antibiotics. Ciprofloxacin (HCI; activity, 84.2%) was obtained from Bayer, Leverkusen, Germany. Stock solutions *
Corresponding author. 2375
were prepared in distilled water and were used within 2 days. Ampicillin (98.8%) was obtained from Gist-Brocades, Delft, The Netherlands. Stock solutions were prepared in phosphate-buffered saline (PBS; pH 7.0) and were used within 1 h. Animals. Female specific-pathogen-free Swiss mice (IFFA Credo, l'Arbresle, France) were used throughout the study. The animals were housed in groups of four in polycarbonate cages on sterile sawdust; they received acidified tap water and nonsterilized food pellets (type AM-II; Hope Farms, Woerden, The Netherlands) ad libitum. Bacteria. An overnight culture of L. monocytogenes EGD in tryptose phosphate broth (Oxoid Ltd., Basingstoke, United Kingdom) was stored in small aliquots at -70°C. The virulence of this strain was maintained by repeated passage through mice. Before each experiment, aliquots were rapidly thawed in a water bath of 37°C. The MIC of ampicillin for this strain was 0.125 mg/liter; that of ciprofloxacin was 1.0 mg/liter. The MICs were determined by the agar dilution method on Iso-Sensitest agar (Oxoid) supplemented with 5% sheep blood. In vitro growth experiments. A 1:3,000 dilution of an overnight culture of L. monocytogenes in tryptose phosphate broth was incubated in a shaking water bath at 37°C for 1 h and then distributed in 20-ml aliqu6ts over 50-ml flasks that contained various concentrations of the antimicrobial drugs. Samples were taken at 1-h intervals over a period of 5 h. After appropriate dilutions in PBS were plated onto blood agar and incubated overnight at 37°C, the CFU was counted. To prevent carryover of the antimicrobial agents, samples expected to have low counts of viable L. monocytogenes cells were washed once with ice-cold PBS. The washing procedure was performed as follows. A 200-,ul sample was diluted with 1,800 ,ul of PBS and was centrifuged at 2,000 x g for 10 min at 4°C; subsequently, the upper 9/10 of the volume was removed with a pipette. Recovery of the bacteria by this procedure was 99.8% + 17.7% (mean ± standard deviation [SD]). Experimental infection model. Mice were treated once
2376
VAN OGTROP ET AL.
daily with 2.5 mg of hydrocortisone acetate (Organon, Oss, The Netherlands) or PBS subcutaneously in the nuchal region. This dose of hydrocortisone has been shown to lead to an increase in the outgrowth of L. monocytogenes in Swiss mice (13). The mice then received an intravenous (i.v.) injection of 1.5 x 103 CFU of L. monocytogenes. The outgrowth of L. monocytogenes in the livers and spleens of hydrocortisone-treated and control mice was determined by quantitative culturing. The initial infection experiments were designed to determine the maximum number of L. monocytogenes cells that could be injected i.v. into hydrocortisone-treated mice which had not received antibiotics. All mice (n = 4) died after an i.v. injection of 2 x 104 CFU of L. monocytogenes between 3 and 4 days after infection, whereas all mice (n = 4) survived for 4 days after an i.v. injection of 2 x 103 CFU of L. monocytogenes. On the basis of the results of these experiments, it was decided that 1.5 x 103 CFU of L. monocytogenes should be used for infection experiments that lasted 4 days and approximately 5.5 x 103 CFU should be used for infection experiments that lasted less than 2 days. At specific intervals after the start of infection, the mice were killed by cervical dislocation, and the livers and spleens were excised, weighed, and homogenized in a tissue homogenizer (Ystral; type X-1020; International Laboratorium Apparate GmbH, Dottingen, Germany). For counting of the CFU in the homogenate, samples were processed as described above for the in vitro experiments. The lower limit of detection for this assay was approximately 50 CFU per organ; when no bacteria were recovered from the organs, the number of CFU was arbitrarily set at 10 for further calculations. Effect of ampicillin and ciprofloxacin on the growth of L. monocytogenes in vivo. The effect of antimicrobial treatment was assessed in hydrocortisone-treated mice. Half an hour after the first injection of hydrocortisone, L. monocytogenes was injected i.v. Twenty-four hours after the injection of L. monocytogenes, the antimicrobial treatment was started. The dose ranges studied were 25 to 200 mg of ampicillin and ciprofloxacin per kg of body weight. The dosage interval in these studies was either 6 or 24 h. Pharmacokinetics. After a single dose of 100 mg of ciprofloxacin or 100 mg of ampicillin per kg, the pharmacokinetics in plasma were determined, each in a group of 22 uninfected mice. At consecutive time points between 15 min and 6 h after administration of the antimicrobial agent, some of the mice were killed by exposure to 100% CO2, and blood samples were taken by cardiac puncture with heparinized syringes and centrifuged at 1,500 x g for 10 min at room temperature and the plasma was removed; the drug concentration was measured as described below. The half-life was then determined by linear regression analysis of the descending slope of the log concentration-time curve, and the area under the concentration-time curve (AUC) was calculated by the trapezoidal method. Protein binding of the antimicrobial agents in murine plasma was determined by equilibrium dialysis in a Dianorm dialysis apparatus (Diachema AG, Zurich, Switzerland) at 37°C (12). Each dialysis chamber has a volume of 1 ml and is separated by a membrane measuring 4.5 cm2. One chamber contained 0.7 ml of plasma, and the other contained a solution of the antibiotic in PBS (pH 7.0). The concentrations studied were 5, 10, and 20 mg/liter for both drugs. The chamber was placed in a rotator, and dialysis was carried out at 16 rpm at 37°C for 4 h, at which time equilibrium was
ANTIMICROB. AGENTS CHEMOTHER. (mg/L)
(mg/L)
0
O0
0.05
0.25
-
I
I. '
0
°B
0.5
0
CD
i1.0 *4.0
68.0 2.0
0
2
1
3
4
5
0
time (hours)
1
2
3
4
5
time (hours)
FIG. 1. In vitro growth of L. monocytogenes in the presence of various concentrations of ampicillin and ciprofloxacin.
obtained. The concentrations of the drugs in both chambers were determined by using appropriate standards. Drug assays. Ciprofloxacin concentrations were determined by an agar diffusion method in a four-point bioassay (11) with a Klebsiella pneumoniae strain as the test organism and nutrient agar (pH 7.4; Oxoid) as the medium. Appropriate twofold dilutions of the samples were prepared with pooled murine plasma. Standards were prepared in the same way, with the twofold steps ranging from 0.25 to 0.063 mg/liter. Ampicillin concentrations were determined by high-performance liquid chromatography as described by Vree et al. (19).
0 L.
U-
61
time (days)
4)
C.)
0
7-
5-
0
2
time (days)
FIG. 2. Outgrowth of L. monocytogenes in the livers and spleens of control mice (circles) and mice receiving 2.5 mg of hydrocortisone acetate subcutaneously once daily (squares). The mice were infected by i.v. injection of 1.5 x 103 CFU of L. monocytogenes at time zero. Each symbol represents the mean log CFU for four mice. The error bars represent SDs.
TREATMENT OF EXPERIMENTAL LISTERIOSIS
VOL. 36, 1992 7-
1-
6-
XI.
0 U. 0
7.-
ampkillin
I
5-
*
ampicillin
6*
0
It
I
m
25
50
100
I
5.-
4
0
2377
200
T
0
25
I
0
10
200
0
25
so
100
200
T
I
100
200
dose (mg/kg)
dose Img/kg)
7-
T
7-
ciprofloxacin
6-
a
U-
ciprotloxacin
6-
b-
e 0
I !
5-
0
25
50
100
:3
200
5.-
0
25
50
dose (mg/kg)
dose (mg/kg)
FIG. 3. Effects of various doses of ampicillin or ciprofloxacin administered subcutaneously on the number of CFU of L. monocytogenes in the livers and spleens of mice treated with hydrocortisone acetate. The mice were infected by i.v. injection of 5.5 x 103 CFU of L. monocytogenes 24 h before administration of the antimicrobial agent. The mice were killed 6 h after administration of the antimicrobial agent. Each symbol represents the mean log CFU for four mice. The error bars represent SDs.
Statistical analysis. The results of assessment of the quantitative cultures are given as the mean log CFU per organ. One-way analysis of variance was used for statistical comparisons. A P level of 0.05 was considered significant.
RESULTS In vitro growth experiments. The exponential growth rate for L. monocytogenes in tryptose phosphate broth, as determined by linear regression analysis, was 0.994 h-1, which corresponds to a doubling time of 44.4 min. Ampicillin showed antibacterial activity at concentrations greater than 0.05 mg/liter. The maximum antibacterial effect of ampicillin on L. monocytogenes was demonstrated at concentrations of 0.1 mg/liter and higher; at these concentrations the effect was bacteriostatic (Fig. 1). Ciprofloxacin showed antibacterial activity at concentrations greater than 0.25 mg/liter (Fig. 1). Concentrations of ciprofloxacin greater than 1 mg/liter led to the killing of L. monocytogenes; the maximum antibacterial effect of ciprofloxacin was found at concentrations of 2 mg/liter and greater.
Experimental infection model. The growth pattern of L. monocytogenes in the livers and spleens of normal and hydrocortisone-treated mice is shown in Fig. 2. The mean SD log number of L. monocytogenes cells in the livers of normal mice increased from 4.31 + 0.15 on day 1 to 6.64 -t --
1.42 on day 4 after infection; for hydrocortisone-treated mice, these numbers were 4.15 + 0.91 on day 1 and 9.03 0.26 on day 4. In the spleens of normal mice the mean + SD log number of L. monocytogenes cells increased from 4.94 +--
0.18 on day 1 to 6.85 + 0.84 on day 4; for hydrocortisonetreated mice, these numbers were 4.71 ± 0.46 on day 1 and 7.68 ± 0.20 on day 4. On day 1, the differences in bacterial counts in the spleens and livers between hydrocortisonetreated mice and normal mice were not statistically significant (P = 0.43 and P = 0.73, respectively), but on day 4 the differences were statistically significant for the livers (P < 0.02) but not for the spleens (P = 0.11). Treatment with hydrocortisone caused a decrease of the average spleen weight from 190 mg in normal mice to 80 mg in hydrocortisone-treated mice at day 4. The differences between hydrocortisone-treated and normal mice were statistically significant for the spleens as well, when the results were expressed as CFU per gram of tissue instead of CFU per organ (P < 0.05). Effect of a single dose of ampicillin or ciprofloxacin on the growth ofL. monocytogenes in vivo. Preliminary experiments showed that after administration of a single subcutaneous dose of 100 mg of ampicillin per kg the number of L. monocytogenes cells in the livers and spleens initially declined but that regrowth of L. monocytogenes occurred after 4 to 6 h. For a single subcutaneous dose of 100 mg of ciprofloxacin per kg, this period was 6 to 8 h. Therefore, a 6-h dosage interval was considered to be the optimum interval for both drugs. In order to determine the dose-effect relationship for ampicillin and ciprofloxacin, the drugs were administered subcutaneously to hydrocortisone-treated mice 24 h after an intravenous injection of 5.5 x 103 CFU of L. monocytogenes. The mice were killed 6 h after administration of the antimicrobial agent. Treatment with ampicillin caused a
2378
VAN OGTROP ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
10
10
-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~b
0
U.o
:.. LL ca
0
CD 0
n
0
time (days) 10
time (days)
-
9c 0
0 0
8-
0
7-
0
6-
0
5
0
2
1
3
time (days)
time (days)
FIG. 4. Effect of 100 mg of ampicillin (squares) or ciprofloxacin (triangles) per kg given subcutaneously once daily on the number of CFU of L. monocytogenes in the livers and spleens of mice treated with hydrocortisone acetate. The mice were infected by i.v. injection of 1.5 x 103 CFU of L. monocytogenes 24 h before the first dose of the antimicrobial agent. Control mice are indicated by circles. Each symbol represents the mean log CFU for four mice. The error bars represent SDs.
FIG. 5. Effect of 100 mg of ampicillin (squares) or ciprofloxacin (triangles) per kg given subcutaneously four times daily on the number of CFU of L. monocytogenes in the livers and spleens of mice treated with hydrocortisone acetate. The mice were infected by i.v. injection of 1.5 x 103 CFU of L. monocytogenes 24 h before the first dose of the antimicrobial agent. Control mice are indicated by circles. Each symbol represents the mean log CFU for four mice. The error bars represent SDs.
significant decrease in the number of L. monocytogenes cells in the livers and spleens, but there was not a significant dose-dependent effect in the dose range between 25 and 200 mg/kg (Fig. 3). The mean + SD log number of L. monocytogenes cells in the livers of ampicillin-treated mice 6 h after administration of the antimicrobial agent was 5.15 ± 0.47, whereas it was 5.91 + 0.52 for control mice (P < 0.005); for the spleens it was 5.09 + 0.31 compared with 5.90 t 0.68 for control mice (P < 0.001). Treatment with 25 to 100 mg of ciprofloxacin per kg had a clear dose-dependent effect (Fig. 3). Six hours after the administration of 100 mg of ciprofloxacin per kg, the mean + SD log number of L. monocytogenes cells in the livers was 5.20 -+- 0.43, whereas it was 6.30 + 0.30 for the control mice (P < 0.001); for the spleens it was 5.28 + 0.24 compared with 6.40 + 0.26 for control mice (P < 0.001). Increasing the dose of ciprofloxacin to 200 mg/kg did not result in a significant increase in the effect (Fig. 3). Effect of 3 days of ampicillin or ciprofloxacin therapy on the growth of L. monocytogenes in vivo. Treatment with a single daily dose of ampicillin or ciprofloxacin led to a relatively small decrease in the number of L. monocytogenes cells in the livers and spleens (Fig. 4). There was no significant difference between ampicillin-treated and ciprofloxacintreated mice in the number of L. monocytogenes cells in the livers and spleens, although on day 3 ciprofloxacin seemed to have had a slightly better effect than ampicillin. Treatment with the antibiotics four times daily led to a much larger reduction in the number of L. monocytogenes cells in the livers and spleens. Treatment with 100 mg of
ampicillin per kg four times daily resulted in a significant decrease (P < 0.001) in the number of L. monocytogenes cells after 1 day of treatment (Fig. 5). Between days 1 and 2 a small, unexpected rise in numbers occurred, but at day 3 the numbers of L. monocytogenes cells again declined to below the detection limit (50 CFU per organ). Treatment with 100 mg of ciprofloxacin per kg also led to a significant decrease (P < 0.001) in the number of L. monocytogenes cells in the livers and spleens, but the decrease in both organs was significantly (P < 0.001) less than that after treatment with ampicillin. In fact, the number of L. monocytogenes cells increased in the livers and spleens of ciprofloxacin-treated mice between days 2 and 3 (Fig. 5). The mean + SD log of the numbers of CFU of L. monocytogenes cells in the livers and spleens of ciprofloxacin-treated mice on day 3 were 4.70 ± 0.19 and 5.02 ± 0.79, respectively, whereas they were 8.25 ± 1.28 and 7.67 ± 0.32, respectively, for the controls. Pharmacokinetic studies. The pharmacokinetic data on a subcutaneous dose of 100 mg of ampicillin or ciprofloxacin per kg in the plasma of mice are given in Table 1. Ampicillin reached a peak concentration in plasma 20 min after injection and had a relatively short half-life in plasma. Ciprofloxacin reached a peak concentration in plasma between 1 and 2 h after injection and had a relatively long half-life in plasma. The AUC of ampicillin from 0 to 6 h was approximately twice as great as the AUC of ciprofloxacin from 0 to 6 h, probably because the apparent clearance of ciprofloxacin from plasma was higher than that of ampicillin. Binding of ampicillin to murine plasma protein amounted to 3%, whereas 10% of ciprofloxacin was plasma protein bound.
TREATMENT OF EXPERIMENTAL LISTERIOSIS
VOL. 36, 1992 TABLE 1. Pharmacokinetics of ampicillin and ciprofloxacin in mouse plasmae Antimicrobial agent
Ampicillin Ciprofloxacin a
Dose . AUCO6 Cm., % Unbound (mg/kg) (mg * h/liter) t1a (min) (mg/liter) drug
100 100
73.5 31.2
23.1 44.1
109.0 12.7
97 90
AUC,_6 area under the concentration-time curve from 0 to 6 h for plasma;
tln, apparent elimination half-life; C,m,, peak concentration in plasma. DISCUSSION
Results of the present study show that ampicillin and ciprofloxacin exhibit antibacterial activities in an experimental Listeria infection in mice immunocompromised by treatment with glucocorticosteroids and that the antibacterial effect of ampicillin is superior to that of ciprofloxacin. These results can be explained by the finding that ampicillin was more potent in vitro than ciprofloxacin against the strain of L. monocytogenes studied and that the in vivo concentrations of ampicillin were much higher than those of ciprofloxacin. However, high concentrations of ciprofloxacin were bactericidal in vitro, whereas ampicillin was merely bacteriostatic. It has been shown that ciprofloxacin penetrates easily into phagocytes (5, 7, 8), while ampicillin penetrates more slowly (9, 18). Apparently, high intracellular concentrations of ampicillin are not essential for the antimicrobial effect against L. monocytogenes in vivo, or alternatively, high intracellular concentrations of ciprofloxacin do not add to the antimicrobial effect against this microorganism. Since it has been shown that L. monocytogenes migrates from macrophages into hepatocytes in nonimmune mice (14), it might well be that ampicillin affects this process more effectively than ciprofloxacin does. Furthermore, it has recently been reported (2) that ampicillin acts synergistically with lysozyme and beta-lysin on the killing of L. monocytogenes, which could explain why ampicillin is more effective than ciprofloxacin in vivo. Glucocorticosteroids exert various effects on the cellular immune response, and therefore, these drugs can inhibit the immune response against L. monocytogenes in several ways. First, glucocorticosteroids have been shown to inhibit the antimicrobial activity of resident macrophages against L. monocytogenes (15). Furthermore, treatment with glucocorticosteroids causes lymphocytopenia (6, 10) and monocytopenia (16). A decrease in the number of blood monocytes reduces the influx of monocytes to the infected organs, while lymphocytopenia leads to suboptimal activation of macrophages because of the decrease in T-helper lymphocytes that produce and secrete lymphokines, such as gamma interferon, which are essential for this activation process. An unexpected result was that after 3 days of treatment with ampicillin, virtually all L. monocytogenes organisms were eliminated from the livers and spleens of glucocorticosteroid-treated mice, because Bakker-Woudenberg et al. (3) found in nude mice, which are also immunocompromised because they lack T lymphocytes, that treatment with ampicillin (in doses of 50 mg per mouse twice daily for 4 days) could not eliminate L. monocytogenes from the livers and spleens. However, there is a difference in the immunological defect between nude mice and glucocorticosteroid-treated mice. For example, the number of L. monocytogenes cells in the organs of nude mice hardly changed during the period of infection and none of the mice died, whereas the number of
2379
L. monocytogenes in the livers and spleens of glucocorticosteroid-treated mice increased gradually. Apparently, nude mice exhibit some degree of host resistance against L. monocytogenes which is lacking in glucocorticosteroidtreated mice. The failure of ampicillin to eliminate L. monocytogenes in nude mnice can be explained by the lack of an effect of this antibiotic on a population of nongrowing bacteria, a well-known characteristic of 3-lactam antibiotics (17). Results of the present study show that the infection model of L. monocytogenes in hydrocortisone-treated mice is suitable for studying the effect of antimicrobial treatment of listeriosis. This model was developed because treatment with glucocorticosteroids renders these mice more susceptible to an i.v. challenge with L. monocytogenes than it does untreated mice (13) and because it was assumed that treatment of mice with glucocorticosteroids would produce a relevant animal model of human listeriosis, a disease which occurs as an opportunistic infection in immunocompromised patients, e.g., patients on glucocorticosteroid therapy (1). ACKNOWVLEDGMENT This work was supported by the J. A. Cohen Institute for Radiopathology and Radiation Protection. REFERENCES 1. Armstrong, D. 1990. Listeria monocytogenes, p. 1587-1593. In G. L. Mandell, R. G. Douglas, and J. E. Bennett (ed.), Principles and practice of infectious diseases, 3rd ed. Churchill Livingstone, New York. 2. Asensi, V., and J. Fierer. 1991. Synergistic effect of human lysozyme plus ampicillin or j-lysin on the killing of Listeria
monocytogens. J. Infect. Dis. 163:574-578. 3. Bakker-Woudenberg, I. A. J. M., P. de Bos, W. B. van Leeuwen, and M. F. Michel. 1981. Efficacy of ampicillin therapy in experimental listeriosis in mice with impaired T-cell-mediated immune response. Antimicrob. Agents Chemother. 19:76-81. 4. Campoli-Richards, D. M., J. P. Monk, A. Price, P. Benfield, P. A. Todd, and A. Ward. 1988. Ciprofloxacin: a review of its antibacterial activity, pharmacokinetic properties and therapeutic use. Drugs 35:373-447. 5. Carlier, M.-B., B. Scorneaux, A. Zenebergh, J.-F. Desnottes, and P. M. Tulkens. 1990. Cellular uptake, localization and activity of fluoroquinolones in uninfected and infected macrophages. J. Antimicrob. Chemother. 26(Suppl. B):27-39. 6. Dougherty, T. F., and A. White. 1943. Influence of adrenal cortical secretion on blood elements. Science 98:367-369. 7. Easmon, C. S. F., and J. P. Crane. 1985. Uptake of ciprofloxacin by human neutrophils. J. Antimicrob. Chemother. 16:67-73. 8. Easmon, C. S. F., and J. P. Crane. 1985. Uptake of ciprofloxacin by macrophages. J. Clin. Pathol. 38:442-444. 9. Gerding, D. N., L. R. Peterson, C. E. Hughes, and D. M. Bamberger. 1986. Extravascular antimicrobial distribution in man, p. 938-994. In V. Lorian (ed.), Antibiotics in laboratory medicine, 2nd ed. The Williams & Wilkins Co., Baltimore. 10. Kowalski, H. J., W. E. Reynolds, and D. D. Rutstein. 1952. Changes in white blood cell counts after administration of cortisone acetate to healthy ambulatory individuals. J. Lab. Clin. Med. 40:841-850. 11. Mattie, H., W. R. 0. Goslings, and E. L. Noach. 1973. Cloxacillin and nafcillin: serum binding and its relationship to antibacterial effect in mice. J. Infect. Dis. 128:170-177. 12. Mattie, H., and G. B. van der Voet. 1981. The relative potency of amoxycillin and ampicillin in vitro and in vivo. Scand. J. Infect. Dis. 13:291-296. 13. North, R. J. 1971. The action of cortisone acetate on cellmediated immunity to infection. J. Exp. Med. 134:1485-1500. 14. Portnoy, D. A., T. Chakraborty, W. Goebel, and P. Cossart. 1992. Molecular determinants of Listeria monocytogenes pathogenesis. Infect. Immun. 60:1263-1267.
2380
VAN OGTROP ET AL.
15. Schaffner, A., and T. Schaffer. 1987. Glucocorticoid-induced impairment of macrophage antimicrobial activity: mechanisms and dependence on the state of activation. Rev. Infect. Dis. 9(Suppl. 5):S620-S629. 16. Thompson, J., and R. van Furth. 1970. The effect of glucocorticosteroids on the kinetics of mononuclear phagocytes. J. Exp. Med. 131:429-442. 17. Tuomanen, E. 1986. Phenotypic tolerance: the search for P-lac-
ANTIMICROB. AGENTS CHEMOTHER.
tam antibiotics that kill nongrowing bacteria. Rev. Infect. Dis. 8(Suppl. 3):S279-S291. 18. Van den Broek, P. J. 1989. Antimicrobial drugs, microorganisms, and phagocytes. Rev. Infect. Dis. 11:213-245. 19. Vree, T. B., Y. A. Hekster, A. M. Baars, and E. van der KleUn. 1978. Rapid determination of amoxycillin and ampicillin in body fluids of man by means of high-performance liquid chromatography. J. Chromatogr. 145:496-501.
0066-4804/92/112375-06$02.00/0 Copyright © 1992, American Society for Microbiology
Vol. 36, No. 11
Comparison of the Antibacterial Efficacies of Ampicillin and Ciprofloxacin against Experimental Infections with Listeria monocytogenes in Hydrocortisone-Treated Mice MARC L. vAN OGTROP,1'2 HERMAN MATTIE,1 BEATA RAZAB SEKH,1 ELISABETH vAN STRIJEN,1 AND RALPH VAN FURTH'* Department of Infectious Diseases, University Hospital, 1 and J. A. Cohen Institute for Radiopathology and Radiation Protection, 2 Leiden, The Netherlands Received 3 February 1992/Accepted 12 August 1992
The efficacies of ciprofloxacin and ampicillin against Listeria monocytogenes in an immunosuppressed mouse model of listeriosis were compared. Immunosuppression was achieved by administration of 2.5 mg of hydrocortisone acetate daily. Both ciprofloxacin and ampicillin were effective in reducing the number of viable L. monocytogenes cells in the liver and spleen. After treatment with 100 mg of ampicillin per kg of body weight every 6 h for 3 days, virtually no L. monocytogenes could be recovered from the livers and spleens of the mice. In contrast, after treatment with 100 mg of ciprofloxacin per kg every 6 h for 3 days, a geometric mean of 5 x 10 CFU of L. monocytogenes was recovered from the spleens and 1 x 10i CFU was recovered from the livers of the mice. Results of the study show that the antibacterial efficacy of ampicillin is far superior to that of ciprofloxacin in our animal model of listeriosis.
Listeria monocytogenes is a facultative intracellular bacterial pathogen that causes severe infections, such as septicemia and meningitis, which can be life threatening in immunocompromised patients. The patients most often affected by severe listeriosis are neonates, transplant recipients, patients on glucocorticosteroids, and patients with lymphomas (1). Standard therapy for listeriosis consists of either ampicillin or penicillin, sometimes combined with an aminoglycoside (1); there are few well-documented alternatives for the treatment of listeriosis. Co-trimoxazole, vancomycin, tetracycline, chloramphenicol, and cephalothin have all been suggested as alternatives to ampicillin (1), but controlled studies have not been performed. Ciprofloxacin is a fluorinated quinolone with in vitro bactericidal activity against L. monocytogenes (4); moreover, it easily penetrates phagocytic cells (5, 7, 8). These characteristics make ciprofloxacin an interesting alternative therapy for listeriosis. In the present study, the antimicrobial efficacy of ciprofloxacin was compared with that of ampicillin for the treatment of an experimental infection with L. monocytogenes in mice immunocompromised by treatment with glucocorticosteroids. The design of the study was, first, to determine the dosage interval for the treatment of this experimental infection by using a maximally effective dose of ampicillin or ciprofloxacin, then to determine the maximally effective dose of either drug after a single dose, and finally, to determine the antibacterial efficacy of both antimicrobial drugs by giving the maximally effective doses at the determined dosage intervals for a period of 3 days. (This study was presented in part at the 5th European Congress of Clinical Microbiology and Infectious Diseases, Oslo, Norway, 1991.)
MATERIALS AND METHODS Antibiotics. Ciprofloxacin (HCI; activity, 84.2%) was obtained from Bayer, Leverkusen, Germany. Stock solutions *
Corresponding author. 2375
were prepared in distilled water and were used within 2 days. Ampicillin (98.8%) was obtained from Gist-Brocades, Delft, The Netherlands. Stock solutions were prepared in phosphate-buffered saline (PBS; pH 7.0) and were used within 1 h. Animals. Female specific-pathogen-free Swiss mice (IFFA Credo, l'Arbresle, France) were used throughout the study. The animals were housed in groups of four in polycarbonate cages on sterile sawdust; they received acidified tap water and nonsterilized food pellets (type AM-II; Hope Farms, Woerden, The Netherlands) ad libitum. Bacteria. An overnight culture of L. monocytogenes EGD in tryptose phosphate broth (Oxoid Ltd., Basingstoke, United Kingdom) was stored in small aliquots at -70°C. The virulence of this strain was maintained by repeated passage through mice. Before each experiment, aliquots were rapidly thawed in a water bath of 37°C. The MIC of ampicillin for this strain was 0.125 mg/liter; that of ciprofloxacin was 1.0 mg/liter. The MICs were determined by the agar dilution method on Iso-Sensitest agar (Oxoid) supplemented with 5% sheep blood. In vitro growth experiments. A 1:3,000 dilution of an overnight culture of L. monocytogenes in tryptose phosphate broth was incubated in a shaking water bath at 37°C for 1 h and then distributed in 20-ml aliqu6ts over 50-ml flasks that contained various concentrations of the antimicrobial drugs. Samples were taken at 1-h intervals over a period of 5 h. After appropriate dilutions in PBS were plated onto blood agar and incubated overnight at 37°C, the CFU was counted. To prevent carryover of the antimicrobial agents, samples expected to have low counts of viable L. monocytogenes cells were washed once with ice-cold PBS. The washing procedure was performed as follows. A 200-,ul sample was diluted with 1,800 ,ul of PBS and was centrifuged at 2,000 x g for 10 min at 4°C; subsequently, the upper 9/10 of the volume was removed with a pipette. Recovery of the bacteria by this procedure was 99.8% + 17.7% (mean ± standard deviation [SD]). Experimental infection model. Mice were treated once
2376
VAN OGTROP ET AL.
daily with 2.5 mg of hydrocortisone acetate (Organon, Oss, The Netherlands) or PBS subcutaneously in the nuchal region. This dose of hydrocortisone has been shown to lead to an increase in the outgrowth of L. monocytogenes in Swiss mice (13). The mice then received an intravenous (i.v.) injection of 1.5 x 103 CFU of L. monocytogenes. The outgrowth of L. monocytogenes in the livers and spleens of hydrocortisone-treated and control mice was determined by quantitative culturing. The initial infection experiments were designed to determine the maximum number of L. monocytogenes cells that could be injected i.v. into hydrocortisone-treated mice which had not received antibiotics. All mice (n = 4) died after an i.v. injection of 2 x 104 CFU of L. monocytogenes between 3 and 4 days after infection, whereas all mice (n = 4) survived for 4 days after an i.v. injection of 2 x 103 CFU of L. monocytogenes. On the basis of the results of these experiments, it was decided that 1.5 x 103 CFU of L. monocytogenes should be used for infection experiments that lasted 4 days and approximately 5.5 x 103 CFU should be used for infection experiments that lasted less than 2 days. At specific intervals after the start of infection, the mice were killed by cervical dislocation, and the livers and spleens were excised, weighed, and homogenized in a tissue homogenizer (Ystral; type X-1020; International Laboratorium Apparate GmbH, Dottingen, Germany). For counting of the CFU in the homogenate, samples were processed as described above for the in vitro experiments. The lower limit of detection for this assay was approximately 50 CFU per organ; when no bacteria were recovered from the organs, the number of CFU was arbitrarily set at 10 for further calculations. Effect of ampicillin and ciprofloxacin on the growth of L. monocytogenes in vivo. The effect of antimicrobial treatment was assessed in hydrocortisone-treated mice. Half an hour after the first injection of hydrocortisone, L. monocytogenes was injected i.v. Twenty-four hours after the injection of L. monocytogenes, the antimicrobial treatment was started. The dose ranges studied were 25 to 200 mg of ampicillin and ciprofloxacin per kg of body weight. The dosage interval in these studies was either 6 or 24 h. Pharmacokinetics. After a single dose of 100 mg of ciprofloxacin or 100 mg of ampicillin per kg, the pharmacokinetics in plasma were determined, each in a group of 22 uninfected mice. At consecutive time points between 15 min and 6 h after administration of the antimicrobial agent, some of the mice were killed by exposure to 100% CO2, and blood samples were taken by cardiac puncture with heparinized syringes and centrifuged at 1,500 x g for 10 min at room temperature and the plasma was removed; the drug concentration was measured as described below. The half-life was then determined by linear regression analysis of the descending slope of the log concentration-time curve, and the area under the concentration-time curve (AUC) was calculated by the trapezoidal method. Protein binding of the antimicrobial agents in murine plasma was determined by equilibrium dialysis in a Dianorm dialysis apparatus (Diachema AG, Zurich, Switzerland) at 37°C (12). Each dialysis chamber has a volume of 1 ml and is separated by a membrane measuring 4.5 cm2. One chamber contained 0.7 ml of plasma, and the other contained a solution of the antibiotic in PBS (pH 7.0). The concentrations studied were 5, 10, and 20 mg/liter for both drugs. The chamber was placed in a rotator, and dialysis was carried out at 16 rpm at 37°C for 4 h, at which time equilibrium was
ANTIMICROB. AGENTS CHEMOTHER. (mg/L)
(mg/L)
0
O0
0.05
0.25
-
I
I. '
0
°B
0.5
0
CD
i1.0 *4.0
68.0 2.0
0
2
1
3
4
5
0
time (hours)
1
2
3
4
5
time (hours)
FIG. 1. In vitro growth of L. monocytogenes in the presence of various concentrations of ampicillin and ciprofloxacin.
obtained. The concentrations of the drugs in both chambers were determined by using appropriate standards. Drug assays. Ciprofloxacin concentrations were determined by an agar diffusion method in a four-point bioassay (11) with a Klebsiella pneumoniae strain as the test organism and nutrient agar (pH 7.4; Oxoid) as the medium. Appropriate twofold dilutions of the samples were prepared with pooled murine plasma. Standards were prepared in the same way, with the twofold steps ranging from 0.25 to 0.063 mg/liter. Ampicillin concentrations were determined by high-performance liquid chromatography as described by Vree et al. (19).
0 L.
U-
61
time (days)
4)
C.)
0
7-
5-
0
2
time (days)
FIG. 2. Outgrowth of L. monocytogenes in the livers and spleens of control mice (circles) and mice receiving 2.5 mg of hydrocortisone acetate subcutaneously once daily (squares). The mice were infected by i.v. injection of 1.5 x 103 CFU of L. monocytogenes at time zero. Each symbol represents the mean log CFU for four mice. The error bars represent SDs.
TREATMENT OF EXPERIMENTAL LISTERIOSIS
VOL. 36, 1992 7-
1-
6-
XI.
0 U. 0
7.-
ampkillin
I
5-
*
ampicillin
6*
0
It
I
m
25
50
100
I
5.-
4
0
2377
200
T
0
25
I
0
10
200
0
25
so
100
200
T
I
100
200
dose (mg/kg)
dose Img/kg)
7-
T
7-
ciprofloxacin
6-
a
U-
ciprotloxacin
6-
b-
e 0
I !
5-
0
25
50
100
:3
200
5.-
0
25
50
dose (mg/kg)
dose (mg/kg)
FIG. 3. Effects of various doses of ampicillin or ciprofloxacin administered subcutaneously on the number of CFU of L. monocytogenes in the livers and spleens of mice treated with hydrocortisone acetate. The mice were infected by i.v. injection of 5.5 x 103 CFU of L. monocytogenes 24 h before administration of the antimicrobial agent. The mice were killed 6 h after administration of the antimicrobial agent. Each symbol represents the mean log CFU for four mice. The error bars represent SDs.
Statistical analysis. The results of assessment of the quantitative cultures are given as the mean log CFU per organ. One-way analysis of variance was used for statistical comparisons. A P level of 0.05 was considered significant.
RESULTS In vitro growth experiments. The exponential growth rate for L. monocytogenes in tryptose phosphate broth, as determined by linear regression analysis, was 0.994 h-1, which corresponds to a doubling time of 44.4 min. Ampicillin showed antibacterial activity at concentrations greater than 0.05 mg/liter. The maximum antibacterial effect of ampicillin on L. monocytogenes was demonstrated at concentrations of 0.1 mg/liter and higher; at these concentrations the effect was bacteriostatic (Fig. 1). Ciprofloxacin showed antibacterial activity at concentrations greater than 0.25 mg/liter (Fig. 1). Concentrations of ciprofloxacin greater than 1 mg/liter led to the killing of L. monocytogenes; the maximum antibacterial effect of ciprofloxacin was found at concentrations of 2 mg/liter and greater.
Experimental infection model. The growth pattern of L. monocytogenes in the livers and spleens of normal and hydrocortisone-treated mice is shown in Fig. 2. The mean SD log number of L. monocytogenes cells in the livers of normal mice increased from 4.31 + 0.15 on day 1 to 6.64 -t --
1.42 on day 4 after infection; for hydrocortisone-treated mice, these numbers were 4.15 + 0.91 on day 1 and 9.03 0.26 on day 4. In the spleens of normal mice the mean + SD log number of L. monocytogenes cells increased from 4.94 +--
0.18 on day 1 to 6.85 + 0.84 on day 4; for hydrocortisonetreated mice, these numbers were 4.71 ± 0.46 on day 1 and 7.68 ± 0.20 on day 4. On day 1, the differences in bacterial counts in the spleens and livers between hydrocortisonetreated mice and normal mice were not statistically significant (P = 0.43 and P = 0.73, respectively), but on day 4 the differences were statistically significant for the livers (P < 0.02) but not for the spleens (P = 0.11). Treatment with hydrocortisone caused a decrease of the average spleen weight from 190 mg in normal mice to 80 mg in hydrocortisone-treated mice at day 4. The differences between hydrocortisone-treated and normal mice were statistically significant for the spleens as well, when the results were expressed as CFU per gram of tissue instead of CFU per organ (P < 0.05). Effect of a single dose of ampicillin or ciprofloxacin on the growth ofL. monocytogenes in vivo. Preliminary experiments showed that after administration of a single subcutaneous dose of 100 mg of ampicillin per kg the number of L. monocytogenes cells in the livers and spleens initially declined but that regrowth of L. monocytogenes occurred after 4 to 6 h. For a single subcutaneous dose of 100 mg of ciprofloxacin per kg, this period was 6 to 8 h. Therefore, a 6-h dosage interval was considered to be the optimum interval for both drugs. In order to determine the dose-effect relationship for ampicillin and ciprofloxacin, the drugs were administered subcutaneously to hydrocortisone-treated mice 24 h after an intravenous injection of 5.5 x 103 CFU of L. monocytogenes. The mice were killed 6 h after administration of the antimicrobial agent. Treatment with ampicillin caused a
2378
VAN OGTROP ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
10
10
-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~b
0
U.o
:.. LL ca
0
CD 0
n
0
time (days) 10
time (days)
-
9c 0
0 0
8-
0
7-
0
6-
0
5
0
2
1
3
time (days)
time (days)
FIG. 4. Effect of 100 mg of ampicillin (squares) or ciprofloxacin (triangles) per kg given subcutaneously once daily on the number of CFU of L. monocytogenes in the livers and spleens of mice treated with hydrocortisone acetate. The mice were infected by i.v. injection of 1.5 x 103 CFU of L. monocytogenes 24 h before the first dose of the antimicrobial agent. Control mice are indicated by circles. Each symbol represents the mean log CFU for four mice. The error bars represent SDs.
FIG. 5. Effect of 100 mg of ampicillin (squares) or ciprofloxacin (triangles) per kg given subcutaneously four times daily on the number of CFU of L. monocytogenes in the livers and spleens of mice treated with hydrocortisone acetate. The mice were infected by i.v. injection of 1.5 x 103 CFU of L. monocytogenes 24 h before the first dose of the antimicrobial agent. Control mice are indicated by circles. Each symbol represents the mean log CFU for four mice. The error bars represent SDs.
significant decrease in the number of L. monocytogenes cells in the livers and spleens, but there was not a significant dose-dependent effect in the dose range between 25 and 200 mg/kg (Fig. 3). The mean + SD log number of L. monocytogenes cells in the livers of ampicillin-treated mice 6 h after administration of the antimicrobial agent was 5.15 ± 0.47, whereas it was 5.91 + 0.52 for control mice (P < 0.005); for the spleens it was 5.09 + 0.31 compared with 5.90 t 0.68 for control mice (P < 0.001). Treatment with 25 to 100 mg of ciprofloxacin per kg had a clear dose-dependent effect (Fig. 3). Six hours after the administration of 100 mg of ciprofloxacin per kg, the mean + SD log number of L. monocytogenes cells in the livers was 5.20 -+- 0.43, whereas it was 6.30 + 0.30 for the control mice (P < 0.001); for the spleens it was 5.28 + 0.24 compared with 6.40 + 0.26 for control mice (P < 0.001). Increasing the dose of ciprofloxacin to 200 mg/kg did not result in a significant increase in the effect (Fig. 3). Effect of 3 days of ampicillin or ciprofloxacin therapy on the growth of L. monocytogenes in vivo. Treatment with a single daily dose of ampicillin or ciprofloxacin led to a relatively small decrease in the number of L. monocytogenes cells in the livers and spleens (Fig. 4). There was no significant difference between ampicillin-treated and ciprofloxacintreated mice in the number of L. monocytogenes cells in the livers and spleens, although on day 3 ciprofloxacin seemed to have had a slightly better effect than ampicillin. Treatment with the antibiotics four times daily led to a much larger reduction in the number of L. monocytogenes cells in the livers and spleens. Treatment with 100 mg of
ampicillin per kg four times daily resulted in a significant decrease (P < 0.001) in the number of L. monocytogenes cells after 1 day of treatment (Fig. 5). Between days 1 and 2 a small, unexpected rise in numbers occurred, but at day 3 the numbers of L. monocytogenes cells again declined to below the detection limit (50 CFU per organ). Treatment with 100 mg of ciprofloxacin per kg also led to a significant decrease (P < 0.001) in the number of L. monocytogenes cells in the livers and spleens, but the decrease in both organs was significantly (P < 0.001) less than that after treatment with ampicillin. In fact, the number of L. monocytogenes cells increased in the livers and spleens of ciprofloxacin-treated mice between days 2 and 3 (Fig. 5). The mean + SD log of the numbers of CFU of L. monocytogenes cells in the livers and spleens of ciprofloxacin-treated mice on day 3 were 4.70 ± 0.19 and 5.02 ± 0.79, respectively, whereas they were 8.25 ± 1.28 and 7.67 ± 0.32, respectively, for the controls. Pharmacokinetic studies. The pharmacokinetic data on a subcutaneous dose of 100 mg of ampicillin or ciprofloxacin per kg in the plasma of mice are given in Table 1. Ampicillin reached a peak concentration in plasma 20 min after injection and had a relatively short half-life in plasma. Ciprofloxacin reached a peak concentration in plasma between 1 and 2 h after injection and had a relatively long half-life in plasma. The AUC of ampicillin from 0 to 6 h was approximately twice as great as the AUC of ciprofloxacin from 0 to 6 h, probably because the apparent clearance of ciprofloxacin from plasma was higher than that of ampicillin. Binding of ampicillin to murine plasma protein amounted to 3%, whereas 10% of ciprofloxacin was plasma protein bound.
TREATMENT OF EXPERIMENTAL LISTERIOSIS
VOL. 36, 1992 TABLE 1. Pharmacokinetics of ampicillin and ciprofloxacin in mouse plasmae Antimicrobial agent
Ampicillin Ciprofloxacin a
Dose . AUCO6 Cm., % Unbound (mg/kg) (mg * h/liter) t1a (min) (mg/liter) drug
100 100
73.5 31.2
23.1 44.1
109.0 12.7
97 90
AUC,_6 area under the concentration-time curve from 0 to 6 h for plasma;
tln, apparent elimination half-life; C,m,, peak concentration in plasma. DISCUSSION
Results of the present study show that ampicillin and ciprofloxacin exhibit antibacterial activities in an experimental Listeria infection in mice immunocompromised by treatment with glucocorticosteroids and that the antibacterial effect of ampicillin is superior to that of ciprofloxacin. These results can be explained by the finding that ampicillin was more potent in vitro than ciprofloxacin against the strain of L. monocytogenes studied and that the in vivo concentrations of ampicillin were much higher than those of ciprofloxacin. However, high concentrations of ciprofloxacin were bactericidal in vitro, whereas ampicillin was merely bacteriostatic. It has been shown that ciprofloxacin penetrates easily into phagocytes (5, 7, 8), while ampicillin penetrates more slowly (9, 18). Apparently, high intracellular concentrations of ampicillin are not essential for the antimicrobial effect against L. monocytogenes in vivo, or alternatively, high intracellular concentrations of ciprofloxacin do not add to the antimicrobial effect against this microorganism. Since it has been shown that L. monocytogenes migrates from macrophages into hepatocytes in nonimmune mice (14), it might well be that ampicillin affects this process more effectively than ciprofloxacin does. Furthermore, it has recently been reported (2) that ampicillin acts synergistically with lysozyme and beta-lysin on the killing of L. monocytogenes, which could explain why ampicillin is more effective than ciprofloxacin in vivo. Glucocorticosteroids exert various effects on the cellular immune response, and therefore, these drugs can inhibit the immune response against L. monocytogenes in several ways. First, glucocorticosteroids have been shown to inhibit the antimicrobial activity of resident macrophages against L. monocytogenes (15). Furthermore, treatment with glucocorticosteroids causes lymphocytopenia (6, 10) and monocytopenia (16). A decrease in the number of blood monocytes reduces the influx of monocytes to the infected organs, while lymphocytopenia leads to suboptimal activation of macrophages because of the decrease in T-helper lymphocytes that produce and secrete lymphokines, such as gamma interferon, which are essential for this activation process. An unexpected result was that after 3 days of treatment with ampicillin, virtually all L. monocytogenes organisms were eliminated from the livers and spleens of glucocorticosteroid-treated mice, because Bakker-Woudenberg et al. (3) found in nude mice, which are also immunocompromised because they lack T lymphocytes, that treatment with ampicillin (in doses of 50 mg per mouse twice daily for 4 days) could not eliminate L. monocytogenes from the livers and spleens. However, there is a difference in the immunological defect between nude mice and glucocorticosteroid-treated mice. For example, the number of L. monocytogenes cells in the organs of nude mice hardly changed during the period of infection and none of the mice died, whereas the number of
2379
L. monocytogenes in the livers and spleens of glucocorticosteroid-treated mice increased gradually. Apparently, nude mice exhibit some degree of host resistance against L. monocytogenes which is lacking in glucocorticosteroidtreated mice. The failure of ampicillin to eliminate L. monocytogenes in nude mnice can be explained by the lack of an effect of this antibiotic on a population of nongrowing bacteria, a well-known characteristic of 3-lactam antibiotics (17). Results of the present study show that the infection model of L. monocytogenes in hydrocortisone-treated mice is suitable for studying the effect of antimicrobial treatment of listeriosis. This model was developed because treatment with glucocorticosteroids renders these mice more susceptible to an i.v. challenge with L. monocytogenes than it does untreated mice (13) and because it was assumed that treatment of mice with glucocorticosteroids would produce a relevant animal model of human listeriosis, a disease which occurs as an opportunistic infection in immunocompromised patients, e.g., patients on glucocorticosteroid therapy (1). ACKNOWVLEDGMENT This work was supported by the J. A. Cohen Institute for Radiopathology and Radiation Protection. REFERENCES 1. Armstrong, D. 1990. Listeria monocytogenes, p. 1587-1593. In G. L. Mandell, R. G. Douglas, and J. E. Bennett (ed.), Principles and practice of infectious diseases, 3rd ed. Churchill Livingstone, New York. 2. Asensi, V., and J. Fierer. 1991. Synergistic effect of human lysozyme plus ampicillin or j-lysin on the killing of Listeria
monocytogens. J. Infect. Dis. 163:574-578. 3. Bakker-Woudenberg, I. A. J. M., P. de Bos, W. B. van Leeuwen, and M. F. Michel. 1981. Efficacy of ampicillin therapy in experimental listeriosis in mice with impaired T-cell-mediated immune response. Antimicrob. Agents Chemother. 19:76-81. 4. Campoli-Richards, D. M., J. P. Monk, A. Price, P. Benfield, P. A. Todd, and A. Ward. 1988. Ciprofloxacin: a review of its antibacterial activity, pharmacokinetic properties and therapeutic use. Drugs 35:373-447. 5. Carlier, M.-B., B. Scorneaux, A. Zenebergh, J.-F. Desnottes, and P. M. Tulkens. 1990. Cellular uptake, localization and activity of fluoroquinolones in uninfected and infected macrophages. J. Antimicrob. Chemother. 26(Suppl. B):27-39. 6. Dougherty, T. F., and A. White. 1943. Influence of adrenal cortical secretion on blood elements. Science 98:367-369. 7. Easmon, C. S. F., and J. P. Crane. 1985. Uptake of ciprofloxacin by human neutrophils. J. Antimicrob. Chemother. 16:67-73. 8. Easmon, C. S. F., and J. P. Crane. 1985. Uptake of ciprofloxacin by macrophages. J. Clin. Pathol. 38:442-444. 9. Gerding, D. N., L. R. Peterson, C. E. Hughes, and D. M. Bamberger. 1986. Extravascular antimicrobial distribution in man, p. 938-994. In V. Lorian (ed.), Antibiotics in laboratory medicine, 2nd ed. The Williams & Wilkins Co., Baltimore. 10. Kowalski, H. J., W. E. Reynolds, and D. D. Rutstein. 1952. Changes in white blood cell counts after administration of cortisone acetate to healthy ambulatory individuals. J. Lab. Clin. Med. 40:841-850. 11. Mattie, H., W. R. 0. Goslings, and E. L. Noach. 1973. Cloxacillin and nafcillin: serum binding and its relationship to antibacterial effect in mice. J. Infect. Dis. 128:170-177. 12. Mattie, H., and G. B. van der Voet. 1981. The relative potency of amoxycillin and ampicillin in vitro and in vivo. Scand. J. Infect. Dis. 13:291-296. 13. North, R. J. 1971. The action of cortisone acetate on cellmediated immunity to infection. J. Exp. Med. 134:1485-1500. 14. Portnoy, D. A., T. Chakraborty, W. Goebel, and P. Cossart. 1992. Molecular determinants of Listeria monocytogenes pathogenesis. Infect. Immun. 60:1263-1267.
2380
VAN OGTROP ET AL.
15. Schaffner, A., and T. Schaffer. 1987. Glucocorticoid-induced impairment of macrophage antimicrobial activity: mechanisms and dependence on the state of activation. Rev. Infect. Dis. 9(Suppl. 5):S620-S629. 16. Thompson, J., and R. van Furth. 1970. The effect of glucocorticosteroids on the kinetics of mononuclear phagocytes. J. Exp. Med. 131:429-442. 17. Tuomanen, E. 1986. Phenotypic tolerance: the search for P-lac-
ANTIMICROB. AGENTS CHEMOTHER.
tam antibiotics that kill nongrowing bacteria. Rev. Infect. Dis. 8(Suppl. 3):S279-S291. 18. Van den Broek, P. J. 1989. Antimicrobial drugs, microorganisms, and phagocytes. Rev. Infect. Dis. 11:213-245. 19. Vree, T. B., Y. A. Hekster, A. M. Baars, and E. van der KleUn. 1978. Rapid determination of amoxycillin and ampicillin in body fluids of man by means of high-performance liquid chromatography. J. Chromatogr. 145:496-501.
