Inhibition of Intracellular Growth of Listeria monocytogenes by Antibiotics

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1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 994, p Vol. 38, No /94/$ Inhibition of Intracellular Growth of Listeria monocytogenes by Antibiotics CHRISTIAN MICHELET,l* JEAN LOUP AVRIL,2 FRANCOIS CARTIER,' AND PATRICK BERCHE3 Clinique des Maladies Infectieuses' and Laboratoire de Bacteriologie-Virologie,2 Centre Hospitalier Regional et Universitaire Rennes, and Laboratoire de Microbiologie, Hopital Necker, Paris, 3 France Received 4 September 993/Returned for modification 4 October 993/Accepted 4 December 993 We studied the activities of antibiotics on the intracellular growth of Listeria monocytogenes in a HeLa cell line. After 24 h of contact with the infected cells, the antibiotics most effective against the intracellular growth of the 0 strains tested were amoxicillin, temafloxacin, and sparfloxacin, which nevertheless failed to totally eliminate the intracellular bacteria. Rifampin and co-trimoxazole had variable effects, depending on the isolates studied. The most active combinations were amoxicillin-sparfloxacin, co-trimoxazole-gentamicin, and sparfloxacin-co-trimoxazole. The results suggest the value of using a cell culture technique to study the activities of antibiotics against certain bacteria with intracellular sites of multiplication. Listeria monocytogenes is a facultative, intracellular, grampositive bacterium that is responsible for severe infections, most of which are transmitted through contaminated food (23, 24), including perinatal infections, septicemia, and meningoencephalitis in humans and a wide variety of animal species (, ). In contrast to the usual in vitro susceptibility of this pathogen to most antibiotics, listeriosis induces a high rate of mortality, ranging from 2 to 44.4% (, 8,, 2). There are several possible reasons for this: (i) the frequent occurrence of listeriosis in immunocompromised patients, including neonates, patients undergoing transplant procedures, and patients with underlying diseases; (ii) the intracellular site of bacterial replication, in which L. monocytogenes is capable of multiplying inside the cytoplasms of many cells, including macrophages, epithelial cells, fibroblasts, and hepatocytes (, 9, ), thus impeding the bactericidal activities of antibiotics in vivo; and (iii) the high incidence of meningoencephalitis, producing severe lesions of the central nervous system, where antibiotic penetration might be restricted. The recommended treatment for listeriosis remains amoxicillin alone or combined with gentamicin, regardless of their apparently weak intracellular diffusion and the absence of synergy between these antibiotics, which has been observed during the course of experimental infections in animals (2). Indeed, the ratio between the intracellular concentration and the extracellular concentration assessed in HeLa cells is 0. for penicillin G and 0.2 for ampicillin (3). Aminoglycosides penetrate HeLa cells very slowly, and after 72 h of incubation, the intracellular concentration/extracellular concentration ratio is 0.9 for gentamicin (3). In view of the high rate of clinical failures observed with the reference treatment, alternative treatments have been proposed, and these alternative treatments have been based on the high degree of intracellular penetration of certain antibiotics. The value of rifampin, certain fluoroquinolones, and even chloramphenicol, alone or in combination, has been emphasized (30). Co-trimoxazole has been proposed for use in patients who are hypersensitive to beta-lactam antibiotics and has also been recommended as first-line treatment because of the good diffusion of sulfame- * Corresponding author. Mailing address: Department of Infectious Diseases, Pontchaillou Hospital, 3033 Rennes Cedex, France. Fax: thoxazole and trimethoprim into the meninges (22, 2). The sporadic nature of listeriosis explains the absence of any controlled clinical trials establishing the superiority of one particular therapeutic protocol. The aim of the work described here was to study the activities of antibiotics on the intracellular growth of L. monocytogenes. This was done by using an in vitro model of intracellular infection with HeLa cells. It was found that there is no correlation between the MICs found during extracellular multiplication in culture media and those observed during intracellular growth of the bacterium. The most active antibiotics under these conditions were amoxicillin, temafloxacin, and sparfloxacin. (Part of the present study was presented at the 32nd Interscience Conference on Antimicrobial Agents and Chemotherapy, Anaheim, Calif., to 4 October 992). MATERIALS AND METHODS Bacterial strains and culture media. The reference strain of L. monocytogenes used first was strain EGD, which is a serotype /2a, hemolysin-producing strain derived from the Institut Trudeau (982) (0). It was grown in tryptic soy broth and was stored at - 80 C in the form of -ml aliquots containing 4.8 x 08 bacteria per ml in the growth phase. Nine wild strains of L. monocytogenes, isolated in 99 and 992 from the blood and cerebrospinal fluid of patients admitted to our institution (Hopital Pontchaillou, Rennes, France), were also used as controls for the main results observed with strain EGD. For each experiment, the aliquots necessary for bacterial inoculation were rapidly thawed and adjusted in minimum essential culture medium (MEM) containing Earle salts (Flow Laboratories, Irvine, Scotland) and supplemented with % essential amino acids (Flow Laboratories) and 2% glutamine (Laboratoire Eurobio, Les Ulis, France); bicarbonate was added to the solution to obtain a constant ph (ph 7.2). Staphylococcus aureus ATCC 2922 was used as the reference strain in in vitro antibiotic susceptibility tests. The identities of the test strains and their serotypes were verified by the Centre National des Listeria (Nantes, France) and were identified in the collection by the following designations: CNL 9492 (serotype /2a), CNL 923 (serotype 4b), CNL 9427 (serotype /2a), CNL 9 (serotype 4b), CNL 922 (serotype 4b), CNL 947 (serotype 4b), CNL 9290 Downloaded from on April, 8 by guest

2 VOL. 38, 994 GROWTH INHIBITION OF INTRACELLULAR L. MONOCYTOGENES 439 (serotype /2b), CNL 903 (serotype /2a), and CNL 90 (serotype /2a). These strains had the same levels of susceptibility as strain EGD to the antibiotics tested by the disk method. Antibiotics. The following antibiotics were provided by the manufacturers in the form of powders suitable for susceptibility testing: penicillin G (Diamant), amoxicillin (Beecham), imipenem cilastatin (Merck), gentamicin (Unilabo), erythromycin (Roussel), azithromycin (Pfizer), clarithromycin and hydroxyclarithromycin (Abbott), tetracycline and chloramphenicol (Sigma), sulfamethoxazole and trimethoprim (Roche), rifampin (Merrel), ciprofloxacin (Bayer), sparfloxacin (Rhone Poulenc), and temafloxacin (Abbott). The concentrations required to study the activities of the antibiotics on intracellular L. monocytogenes and in the culture broth were adjusted on the day of the study by using modified MEM supplemented with 0% fetal calf serum (FCS) (TechGen, Les Ulis, France). The activities of the antibiotics were measured by using antibiotic concentrations of 0.0, 0., 0.,,, 0, and mg/liter for strain EGD and,, and mg/liter for the clinical isolates. The concentrations used to study antibiotic combinations were selected according to the efficacy of each antibiotic in the combination alone and the concentrations usually obtained in human serum (0.,, and 0 mg/liter). MICs and MBCs. MICs were determined by the broth dilution technique on tryptic soy microplates (Falcon; Becton- Dickinson Labware, Lincoln Park, Mich.) with Mueller-Hinton broth (Diagnostics Pasteur, Marnes La Coquette, France) and an inoculum of 0 bacteria in the logarithmic phase of growth per spot. The MIC was defined as the lowest concentration of each antibiotic which, after 8 h of incubation at 37 C, inhibited all bacterial growth visible to the naked eye in each well. The MBC, which was determined after inoculation of 0.0 ml of each well containing no visible growth onto Mueller- Hinton agar with a Steers apparatus, was defined as the lowest concentration of antibiotic which decreased the number of viable bacteria by 99.9%. Bactericidal growth curves. The bactericidal kinetics over 24 h, which were determined for the antibiotics alone and in combination, were determined by using a mean inoculum of 2. x 0 L. monocytogenes per ml (range,.4 x 0 to 3 x 0/ml) in modified MEM supplemented with 0% FCS without hormone. After incubation of the bacteria at 37 C with each concentration of the test antibiotics, the number of bacteria were counted after 2,, and 24 h of contact by spreading onto tryptic soy agar (Diagnostics Pasteur) 0. ml of the solution obtained after diluting the culture 0-fold in distilled water. The limit of sensitivity was defined as at least one dilution (log2 CFU) to avoid any carryover effects. For combinations that included gentamicin, the bacteria were washed after centrifugation at,300 x g for min. For four antibiotics, amoxicillin, penicillin G, rifampin, and co-trimoxazole, the number of bacteria was determined by using the same inoculum in tryptic soy broth containing % glucose. The activities of the antibiotics tested were compared in terms of their 7og, the time necessary to reduce the bacterial population by log unit, and TMB, the time necessary to reduce the bacterial population to 0.0% survivors (7). Maintenance and infection of HeLa cells. After thawing, the HeLa cell line was maintained in modified MEM supplemented with 0% FCS and was cultured in 7-cm3 plastic cell culture plates (Falcon 3024) at 37 C in an atmosphere containing % CO2. Semiconfluent cells were trypsinized and adjusted to a concentration of 2. x 0 to 4 x 0 cells per ml with culture medium. Two milliliters of this suspension was deposited into each of the six 3-mm wells of the cell culture plates (Falcon 304), and the plates were incubated at 37 C in an atmosphere containing % CO2 for 72 h. The semiconfluent monolayer of HeLa cells was then washed twice with phosphate-buffered saline (PBS) before inoculation with L. monocytogenes. The number of infected cells in each experiment was counted and was found to range between 8 x 0 and 2. x 0 per well. The semiconfluent monolayer of HeLa cells was inoculated with a suspension of L. monocytogenes adjusted in MEM (without FCS) in order to obtain a multiplicity of infection of 0 bacteria per cell. After h of incubation at 37 C, the infected cells were washed five times with PBS and were then covered with 2 ml of MEM supplemented with 0% FCS and mg of gentamicin per liter to kill the extracellular bacteria. After 90 min of contact, the cells were again washed five times with PBS, were covered with 2 ml of the test concentration of antibiotic or antibiotic combination, and were incubated for another 24 h. After washing five times in PBS to remove any extracellular antibiotic, the infected cells were lysed in 2 ml of distilled water maintained on crushed ice for min. The viable intracellular bacteria were counted by spreading 0. ml of the solution obtained after 0-fold dilution in distilled water onto tryptic soy agar; they were counted h after infection (T- h), after 90 min of contact with gentamicin (TO), and after 2 h (T2), h (T), and 24 h (T24) of contact with the test antibiotic. A control test without antibiotic was performed each time that the number of viable intracellular bacteria was counted. The activity of each concentration of antibiotic was determined simultaneously in three culture wells (in triplicate). The results were expressed as the mean loglo viable bacteria per well. The most important results were checked by performing in triplicate another experiment at another time under the same conditions (the results presented in Tables 2 and 3 correspond to the mean bacterial counts in six inoculated wells). The infection of HeLa cells by strain EGD was initially verified by 8-h bacterial growth curves with inocula ranging from 0 to 00 bacteria per cell. The intracellular penetration and multiplication of L. monocytogenes in HeLa cells were visualized by light microscopy (immersion objective, x 00) at 0, 2,, and 24 h after May-Grunwald Giemsa staining in the absence of antibiotic and in the presence of amoxicillin. A test of cellular viability was performed at 24 h of infection without antibiotic and in the presence of amoxicillin and azithromycin. After washing and verification of the integrity of the semiconfluent monolayer by light microscopy, this layer was covered with 2 ml of trypan blue (0.2%). The number of cells stained with trypan blue was counted in each well on 0 contiguous cells. RESULTS In vitro susceptibilities of L. monocytogenes to antibiotics. The MICs and MBCs of antibiotics were determined in liquid culture medium for strain EGD (Table ). The MICs of the beta-lactams (penicillin, amoxicillin, and imipenem) were less than or equal to 0.03 mg/liter, while the MBCs of these same antibiotics were estimated to be 2 mg/liter. For most of the other antibiotics tested, the MBCs were several times greater than the MICs. Only ciprofloxacin and co-trimoxazole had MBCs equal to the MICs, but ciprofloxacin and sparfloxacin both had MICs greater than or equal to mg/liter. The in vitro bactericidal effects were also determined for strain EGD in MEM, as reported in Table 2 (single antibiotic) and Table 3 (antibiotic combinations). These results were compared with the bactericidal effects of the antibiotics used at the same dose Downloaded from on April, 8 by guest

3 440 MICHELET ET AL. TABLE. Susceptibility of L. monocytogenes EGD to antibiotics Antibiotic MIC (mg/liter) MBC (mg/liter) Penicillin G < Amoxicillin < Imipenem < Gentamicin Chloramphenicol Erythromycin 0.2 Clarithromycin <0.03 Azithromycin 0.2 Tetracycline < Rifampin 0.2 Co-trimoxazole Ciprofloxacin Sparfloxacin 2 4 Temafloxacin 0. against intracellular bacteria grown in HeLa cells, as expressed by the percentage of bacteria surviving after 24 h of incubation in comparison with the initial inoculum. No difference from the results obtained with the controls was observed at concentrations of 0.0 mg/liter, regardless of the antibiotic tested (data not shown). For the beta-lactams tested (penicillin, amoxicillin, and imipenem), more than 0.% of the bacteria survived, with a log of more than h. An intense dose-dependent effect was observed for imipenem in contrast to that for amoxicillin. Gentamicin was rapidly bactericidal, with a dose-dependent activity and a TMB of 2 h at the highest concentration tested ( mg/liter), while at lower concentrations (0. and mg/liter), the proportions of bacteria surviving after 2 h at the lower concentrations were 0.3 and 0.%, respectively, and did not decrease any further as a function of incubation time. Rifampin, clarithromycin, and azithromycin expressed nondose-dependent, essentially bacteriostatic activities. Chloramphenicol at a concentration of mg/liter also exerted bacteriostatic activity, while tetracycline showed no activity. Co-trimoxazole had poor, non-dose-dependent, slow bactericidal activity in MEM. This activity depended on the culture medium used; in tryptic soy broth, the number of bacteria surviving at 24 h was 00 times less than the number surviving in MEM (0.2%) for concentrations of 0. to mg/liter, and Tlog was equal to h (data not shown). Ciprofloxacin and temafloxacin were bactericidal at the highest concentrations tested ( mg/liter), with a TMB equal to 24 h. Sparfloxacin was more slowly bactericidal, with a Tlog of 24 h. At concentrations of mg/liter ciprofloxacin and temafloxacin were bacteriostatic (4% survivors) and sparfloxacin had no effect. The combinations of amoxicillin-gentamicin and co-trimoxazole-gentamicin were rapidly bactericidal, with a TMB equal to 2 h for combinations containing mg of gentamicin per liter. No synergistic effect between the antibiotics in the combinations was observed. The bactericidal curve for the combination of amoxicillin ( mg/liter) and gentamicin ( mg/liter) was identical to that for gentamicin alone. The amoxicillin-rifampin combination was antagonistic, and the rifampin-cotrimoxazole combination was indifferent. Intracellular activities of the antibiotics against L. monocytogenes. The study of the viability of HeLa cells performed 24 h after infection with L. monocytogenes EGD showed that 99% of cells without antibiotic (controls) were stained by trypan blue. In contrast, almost 00% of cells incubated with amoxicillin or azithromycin remained intact. ANTIMICROB. AGENTS CHEMOTHER. TABLE 2. Comparative activities of the study antibiotics against intracellular and extracellular L. monocytogenes EGD Concn Antibiotic (mgacnter) Amoxicilli'n 0 l 0. Penicillin Imipenem Gentamicin Erythromycin Clarithromycin Hydroxyclarithromycin Azithromycin Rifampin Tetracycline Co-trimoxazole Chloramphenicol Ciprofloxacin Temafloxacin 0. S 0. S S Intracellular bacteriaa Percent Extracellular bacteriab 2c id 2d 0.4d 00 id " ' OC < d 3" 0d d " 0 <0.0 < Sparfloxacin 7 90 NDf a Proportion of bacteria that survived in HeLa cells after 24 h of incubation with various concentrations of the antibiotics tested in relation to the bacterial count at time zero. b Proportion of bacteria that survived after 24 h of incubation with various concentrations of antibiotic in cell-free MEM in relation to the initial inoculum (that at time zero). ' Data correspond to the means of two experiments performed in triplicate at different times. d The experiment also conducted on tryptic soy glucose broth; the results were identical for all antibiotics except co-trimoxazole, for which the bactericidal action was less than % and was not concentration dependent. ', no activity. f ND, not done. Downloaded from on April, 8 by guest

4 VOL. 38, 994 GROWTH INHIBITION OF INTRACELLULAR L. MONOCYTOGENES 44 TABLE 3. Antibiotic activities against intracellular and extracellular L. monocytogenes EGD Antibiotic Concn (mg/liter) of Antibiotic 2 Concn (mg/liter) of Percent antibiotic antibiotic 2 Intracellular Extracellular bacteriaa bacteriab Amoxicillin 0 IC Amoxicillin 0.c Co-trimoxazole 7 Rifampin 2 Sparfloxacin d Azithromycin Amoxicillin 0 Gentamicin 3 < <0.0 3 < Amoxicillin 0 Co-trimoxazole Rifampin Azithromycin 0 Sparfloxacin 3 0 Sparfloxacin 0. Co-trimoxazole Gentamicin.c c <0.0 Rifampin Sparfloxacin 3 4 a b Proportion of bacteria that survived (CFU) in HeLa cells after 24 h of incubation in relation to the bacterial count at time zero. Proportion of bacteria surviving (CFU) after 24 h of incubation in cell-free MEM in relation to the initial inoculum. c Data correspond to the means of two experiments performed in triplicate at different times. d, no activity. Downloaded from Amoxicillin induced a marked decrease in the bacterial inoculum without inducing a complete elimination of L. monocytogenes after 24 h of incubation. The mean proportion of surviving bacteria was 2% at concentrations of and 0 mg/liter (Table 2). This was obtained slowly (80% of survivors after h) and required concentrations equal to 00 times the MIC (Fig. ). At concentrations of and 0 mg/liter, the intracellular activity of amoxicillin was similar to its extracellular activity, while at lower concentrations, amoxicillin became bacteriostatic, nevertheless reflecting penetration of the antibiotic into the cell. The activity of penicillin G was less intense (0% of survivors at a concentration of mg/liter) and was not dose dependent. Imipenem was bactericidal only at the highest concentration tested (4% survivors at mg/liter). Despite the rapid extracellular bactericidal activity of gentamicin, it had little influence on intracellular bacterial growth after 24 h of contact (Fig. ). The macrolides and tetracycline were only bacteriostatic; clarithromycin demonstrated the best activity in this model, even at low concentrations, with identical activities at concentrations ranging between 0. and mg of clarithromycin per liter. Azithromycin and erythromycin were observed to have bacteriostatic activities when they were tested at concentrations of to mg/liter. Chloramphenicol was bactericidal only at the highest concentration tested ( mg/liter). The activity of co-trimoxazole was similar to that of amoxicillin and was only slightly dose dependent (% survivors at a concentration of mg/liter). This action was very slow, because no change in the bacterial count was observed at h of incubation (Fig. ). The actions of the fluoroquinolones varied according to the drug studied, but the maximal action was obtained only with the higher concentrations: no activity at mg/liter and bacteriostatic activity at and mg/liter for ciprofloxacin; bactericidal activity for temafloxacin at and mg/liter (2% survivors; the highest concentration increased the rapidity of this action) (Fig. ); and no activity at mg/liter, bacteriostatic activity at mg/liter, and bactericidal activity at mg/liter for sparfloxacin (7% survivors). We then tested the intracellular activities of the antibiotics against nine strains isolated from humans with Listeria infections. The results were concordant with those obtained with strain EGD, despite a few differences in activity, especially for the macrolides and sparfloxacin. This activity was measured by comparing the bacterial count after 24 h of incubation with the antibiotic with the initial bacterial count, expressed as loglo. Amoxicillin showed intense activity with a dose-dependent effect and variable activity according to the strain: a mean.-log-unit decline in the bacterial count (with a range of activity, according to the isolate of -0.2 to -2 log units) at a concentration of mg/liter and a mean.-log-unit decline in bacterial count (range, -. to -2 log units) at a concentration of mg/liter. on April, 8 by guest

5 442 MICHELET ET AL. ANTIMICROB. AGENT'S CHEMOTHER. J 8. GENTAMICIN 9 -- Y.TM A sssrmwe4 T A J w 0-0 -J 9 ERYTHROMYCIN 9] CLARITROMYCIN 9] AZITHROMYCIN QO A I ' 3 9 7, SPARFLOXACIN. i 7. - s m I I I I /~~~~~~~~.0 RIFAMPICIN Downloaded from 3 RW TIME IN HR 24X o FIG.. Activities of 2 antibiotics at different concentrations on intracellular L. monocytogenes after 24 h of incubation. The bacterial counts were determined at 0, 2,, and 24 h of incubation. Symbols: 0, control; A, 0. mg/liter; 0, 0. mg/liter; OI, mg/liter; +, mg/liter; A, 0 mg/liter; *, mg/liter. on April, 8 by guest The activity of the penicillin G was less intense and was only slightly dose dependent (Table 4). The activity of imipenem was dose dependent; bactericidal activity was observed only at a concentration of mg/liter (mean,.-log-unit decline in bacterial count). The bacteriostatic activities of the macrolides (clarithromycin, azithromycin, and erythromycin) were comparable and were not dose dependent at concentrations greater than or equal to mg/liter. However, a decline in the bacterial count of more than log unit was observed for two isolates with clarithromycin, while no activity was observed against another two isolates with azithromycin. Tetracycline showed a bacteriostatic activity at a concentration of mg/liter (0.3-log-unit decline in the bacterial count, with a range of 0.0 to log - - unit, depending on the isolate). Rifampin demonstrated very variable, non-dose-dependent activity against the various clinical isolates: the decline in bacterial counts ranged from - 0. to -2.7 log units. Identical results were obtained with cotrimoxazole, which demonstrated dose-dependent activity that varied greatly among the different isolates (Table 4). Ciprofloxacin did not exert any activity at concentrations less than or equal to mg/liter. Sparfloxacin showed good bactericidal activity at concentrations of mg/liter (2-log-unit decline in bacterial count) and mg/liter (Table 4). Temafloxacin had moderate bactericidal activity at a concentration of mg/liter (-log-unit decline in the bacterial count) but good bactericidal activity at a concentration of mg/liter (almost a 3-log-unit decline in bacterial count).

6 VOL. 38, 994 GROWTH INHIBITION OF INTRACELLULAR L. MONOCYTOGENES 443 TABLE 4. Comparative activities of antibiotics against nine clinical isolates of L. monocytogenes Logo bacterial decrease" Antibiotic Concn Nine clinical strains Median for (mg/liter) Median for Median Range strain EGD Amoxicillin Penicillin Imipenem b Erythromycin Clarithromycin Azithromycin Rifampin Tetracycline Co-trimoxazole Ciprofloxacin Sparfloxacin Temafloxacin a The results are the differences between the bacterial count determined at time zero (after gentamicin incubation) and the bacterial count determined after 24 h of incubation of the antibiotic, expressed as log0. b, no activity. AMOXYCILLIN GENTAMICIN -COTRIMC 04 *7v 4 ~~~~~~~~4- GENTAMI Intracellular activities of antibiotic combinations on L. monocytogenes. The activities of the antibiotic combinations tested against L. monocytogenes EGD were no better than that of amoxicillin alone (Table 3). The addition of gentamicin to amoxicillin did not alter the kinetics of bacterial decline induced by the action of amoxicillin alone (Fig. 2). The combination of co-trimoxazole-gentamicin was synergistic, and the activity of the combination was comparable to that of amoxicillin (a mean of % survivors at 24 h). The bactericidal effect was observed later, beyond the h of incubation. The bacterial decline obtained with the amoxicillin-cotrimoxazole combination was less intense than that obtained with amoxicillin alone, regardless of the concentration used. The amoxicillin-rifampin combination was antagonistic (Fig. 3), while the amoxicillin-azithromycin combination was indifferent (data not shown). The sparfloxacin-amoxicillin combination (bacterial count decrease, -3 log units) and the sparfloxacin-co-trimoxazole combination (bacterial count decrease, <-2 log units) induced more intense bactericidal actions than the bactericidal action obtained with amoxicillin or co-trimoxazole alone (Fig. 4). DISCUSSION The study described here was conducted in a HeLa cell line infected with virulent strains of L. monocytogenes and demonstrated that sparfloxacin and amoxicillin possess the most potent and most constant bactericidal activities at concentrations compatible with clinical use. Co-trimoxazole alone exerted less intense and less constant activity than amoxicillin, but the combination of co-trimoxazole with gentamicin increased the intracellular activity of co-trimoxazole. Similar results were noted in an experimental infection, in which amoxicillin possessed the best activity and co-trimoxazole was not more effective than amoxicillin (3). Temafloxacin was found to be an active molecule, but the useful concentrations ( mg/liter) were high in relation to the doses generally used to treat humans. The efficacies of the sparfloxacin-amoxicillin and co-trimoxazole-sparfloxacin combinations require further evaluations in an animal model before they can be proposed for clinical use in humans. However, none of the antibiotics Downloaded from on April, 8 by guest FIG. 2. I ** Bacterial activities of amoxicillin, co-trimoxazole, and gentamicin in combinations against intracellular L. monocytogenes. The bacterial counts were determined at 0, 2,, and 24 h of incubation. Symbols: +, control; *, amoxicillin at 0 mg/liter; 0, amoxicillin plus gentamicin at mg/liter; 0, amoxicillin plus gentamicin at mg/liter; A, co-trimoxazole at mg/liter; 0, co-trimoxazole plus gentamicin at mg/liter; 0, co-trimoxazole plus gentamicin at mg/liter; O, amoxicillin plus co-trimoxazole.

7 444 MICHELET ET AL. ANTIMICROB. AGENTS CHEMOTHER. 8,0 0i4 C4,0 4,0 2, FIG. 3. Bacterial activities of amoxicillin, co-trimoxazole, and rifampin in combinations against intracellular L. monocytogenes. The bacterial counts were determined at 0, 2,, and 24 h of incubation. Symbols: +, control; U, amoxicillin at mg/liter; 0, amoxicillin plus rifampin at mg/liter; 0, rifampin at mg/liter; A, co-trimoxazole at mg/liter; A, co-trimoxazole plus rifampin. tested was able to completely eradicate intracellular L. monocytogenes within 24 h. The susceptibilities of strain EGD and the clinical isolates of L. monocytogenes used for the present study were comparable to the susceptibility data reported in the literature (2, ). Gentamicin was rapidly bactericidal against extracellular L. monocytogenes, and its use in patients with bacteremia with multiple visceral involvement appears to be fully justified. The synergy of the amoxicillin-gentamicin combination, which is one of the bacteriological bases for the reference treatment of neonatal listeriosis and Listeria meningitis, was not observed during intracellular infection of HeLa cells, nor was it observed in the experimental animal model of L. monocytogenes infection (2). Imipenem alone or in combination with gentamicin was only weakly bactericidal in an experimental model of L. monocytogenes infections in newborn rats, despite the very low MIC of this drug (4). As described previously (, 2), combinations of rifampin and amoxicillin or rifampin and chloramphenicol are also frequently antagonistic. The results of the present study demonstrate the absence of a correlation between the usual bacteriological data (the MIC, MBC, and bactericidal curves) and the actual efficacy of the antibiotic against an intracellular organism, and the conclusions confirm those obtained in an experimental model of animals infected with L. monocytogenes (3). Cure of Listeria infection is primarily dependent on the host's immune defenses, as demonstrated by the acquisition of immunity after infection with a sublethal dose in mice (7). The role played by the immune system is demonstrated by the marked spontaneous decline in the bacterial count in splenic macrophages after the third day of infection in mice in the absence of immunodepression and by the weak bactericidal activity of amoxicillin in athymic mice (, 2). The failure of reference treatment, amoxicillin alone or in combination with gentamicin, is essentially due to an underlying immune defi- COTRIMOXAZOLE + SPARFLOXACIN Downloaded from on April, 8 by guest FIG Bacterial activities of amoxicillin, co-trimoxazole, and sparfloxacin in combinations against intracellular L. monocytogenes. The bacterial counts were determined at 0, 2,, and 24 h of incubation. Symbols: +, control; *, amoxicillin at mg/liter; A, sparfloxacin at mg/liter; O, sparfloxacin at mg/liter; O, amoxicillin plus sparfloxacin at mg/liter; *, amoxicillin plus sparfloxacin at mg/liter; A, co-trimoxazole at mg/liter; 0, co-trimoxazole plus sparfloxacin at mg/liter; 0, co-trimoxazole plus sparfloxacin at mg/liter.

8 VOL. 38, 994 GROWTH INHIBITION OF INTRACELLULAR L. MONOCYTOGENES 44 ciency (8, ). The same applies to the failure of co-trimoxazole treatment alone or in combination with an aminoglycoside, which has not been found to be any more effective than amoxicillin alone (2), hence the value of using experimental models which take this factor into account. However, the culture medium used may interfere with the activities of certain antibiotics such as co-trimoxazole because of its high thymidine content; co-trimoxazole was bacteriostatic only in supplemented MEM, but it demonstrated bactericidal effects in tryptic soy glucose broth. These findings could account for the underestimation of the activity of co-trimoxazole against L. monocytogenes and should be confirmed in another cell system, such as in macrophages, which are mainly cells in which intracellular multiplication of this bacterium occurs. Infection of athymic mice demonstrated the limits of antibiotic treatment in immunosuppressed animals because of the bacterial regrowth in macrophages as soon as treatment with amoxicillin or rifampicin was stopped (). Clarithromycin (9) and rifampin () were able to eradicate listeria infections in immunocompetent mice after several days of treatment, but in immunosuppressed mice, clarithromycin is only bacteriostatic (). All of these results confirm the difficulty of treating Listeria infections and should encourage investigators to search for new molecules and new therapeutic strategies for the treatment of Listeria infections, such as use of the sparfloxacin-amoxicillin combination. L. monocytogenes infection also represents an excellent model for studying the intracellular behaviors of certain antibiotics. Amoxicillin diffuses only into the cytosol of the macrophage, where it comes into contact with the bacteria in the process of multiplication. This helps to explain its activity, despite a high MBC of 2 mg/liter and poor intracellular penetration (intracellular concentration/extracellular concentration ratio, 0.4) (3). In our model of HeLa cells infected with L. monocytogenes, an increase in the concentration of amoxicillin in the culture medium was accompanied by only a moderate increase in bactericidal activity; there does not appear to be a limiting step to the entry of this drug into HeLa cells (3), but the influx would appear to be minor. Cotrimoxazole, which has a more favorable MBC (0.2 mg/liter), also shows poor intracellular accumulation in HeLa cells (3). It may diffuse exclusively into the cytosol, which would explain its activity against L. monocytogenes. Conversely, antibiotics with the best intracellular accumulation, such as macrolides, tetracyclines, and fluoroquinolones (4,, 28, 29), are only bacteriostatic, with the exception of sparfloxacin, which demonstrated excellent bactericidal activity against clinical isolates in the HeLa cell model of L. monocytogenes infection. The intracellular penetration of aminoglycosides is slow, but it may reach twice the extracellular concentration after 72 h of incubation (3, 27). Aminoglycosides are inactive in the phagolysosome because of the acidic ph of phagolysosomes, but they may be active in the cytosol, which could have clinical implications if this action is confirmed. Our experiments were performed with constant extracellular concentrations of drugs over 24 h, as is the case for most other in vitro tests. This allows for the constant intracellular diffusion of antibiotics, which is different from the in vivo conditions. However, our results for HeLa-infected cells were very similar to those obtained in vivo in an experimental model. In conclusion, the model used in the present study, which consisted of infection of nonprofessional phagocytic HeLa cells, was able to integrate a possible underlying immune deficiency and more accurately assess the intrinsic activities of antibiotics against intracellular L. monocytogenes, especially because the data obtained are very reproducible and similar to those observed in the course of experimental infection in animals. ACKNOWLEDGMENTS We thank Nicolas Vu, laboratory technician, for invaluable assistance with the study, P. Pouedras for collaboration, M. F. Travers, and the Bacteriology-Virology Unit of the Hpital Pontchaillou de Rennes. This study was performed with the financial support of INSERM (CJF9004). REFERENCES. Bille, J., and M. P. Glauser Listeriose en Suisse. Bull. Off. Fed. Sante Publique 3: Boisisvon, A., C. Guiomar, and C. Carbon In vitro bactericidal activity of amoxycillin, gentamicin, rifampicin, ciprofloxacin and trimethoprim-sulfamethoxazole alone or in combination against Listeria monocytogenes. Eur. J. Clin. Microbiol. Infect. Dis. 9: Brown, K. B., and A. Percival Penetration of antimicrobials into tissue culture cells and leucocytes. Scand. J. Infect. Dis. 4(Suppl.): Carlier, M. B., B. Scorneaux, A. Zenebergh, J. F. Desnottes, and P. M. Tulkens Cellular uptake, localization, and activity of fluoroquinolones in uninfected and infected macrophages. J. Antimicrob. Chemother. 2(Suppl.): Carlier, M. B., A. Zenebergh, and P. M. Tulkens Cellular uptake and subcellular distribution of roxithromycin and erythromycin in phagocytic cells. J. Antimicrob. Chemother. (Suppl.): Cossart, P., and J. Mengaud Listeria monocytogenes. A model system for the molecular study of intracellular parasitism. Mol. Biol. Med. : Drugeon, H. B., and A. L. Courtieu Comparaison des pouvoirs bactericides de 4 aminosides: amikacine, gentamicine, kanamycine et tobramycine. Ann. Microbiol. (Inst. Pasteur). 30A: Flemming, D. W., S. L. Cochi, K. L. Macdonald, J. Brondum, P. S. Hayes, B. D. Plikaytisis, M. B. Holmes, et al. 98. Pasteurized milk as a vehicle of infection in an outbreak of listeriosis. N. Engl. J. Med. 32: Gaillard, J. L., P. Berche, J. Mounier, S. Richard, and P. Sansonetti In vitro model of penetration and intracellular growth of Listeria monocytogenes in the human enterocyte-like cell line Caco-2. Infect. Immun. : Gaillard, J. L., P. Berche, and P. Sansonetti. 98. Transposon mutagenesis as a tool to study the role of hemolysin in the virulence of Listeria monocytogenes. Infect. Immun. 2:0-.. Hof, H. 99. Therapeutic activities of antibiotics in listeriosis. Infection 9(Suppl.):S229-S Hof, H., and H. Gfickel Lack of synergism of ampicillin and gentamicin in experimental listeriosis. Infection : Hof, H., and G. Waldenmeier Therapy of experimental listeriosis. An evaluation of different antibiotics. Infection (Suppl.):S7-S Kim, K. S. 98. In vitro and in vivo studies of imipenem-cilastatin alone and in combination with gentamicin against Listeria monocytogenes. Antimicrob. Agents Chemother. 29: Linnan, M. J., M. Mascoli, D. L. Xiao, V. Goulet, S. May, C. Salminen, and D. W. Hird Epidemic listeriosis associated with Mexican-style cheese. N. Engl. J. Med. 39: MacGovan, A. P., H. A. Holt, M. J. Bywter, and D. S. Reeves In vitro antimicrobial susceptibility of Listeria monocytogenes isolated in the U.K. and other Listeria species. Eur. J. Clin. Microbiol. Infect. Dis. 9: Mackaness, G. B. 92. Cellular resistance to infection. J. Exp. Med. : McLauchin, J., A. Audurier, and A. G. Taylor. 99. Treatment failure and recurrent human listeriosis. J. Antimicrob. Chemother. 27: Nichterlein, T., J. Bauer, and H. Hof Effect of clarithromycin on infection with Listeria monocytogenes in mice and in L929 Downloaded from on April, 8 by guest

9 44 MICHELET ET AL. ANTIMICROB. AGENTS CHEMOTHER. cells, p th International Symposium on the Problems of Listeriosis.. Nieman, R. E., and B. Lorber Listeriosis in adults: a changing pattern. Report of eight cases and review of the literature, Rev. Infect. Dis. 2: North, R. J., P. Berche, and M. F. Newborg. 98. Immunologic consequences of antibiotic-induced abridgement of bacterial infection: effect on generation and loss of protective T cells and level of immunologic memory. J. Immunol. 27: Scheld, W. M., D. D. Flectcher, F. N. Fink, and M. A. Sande Response to therapy in an experimental rabbit model of meningitis due to Listeria monocytogenes. J. Infect. Dis. 40: Schlech, W. F., P. M. Lavigne, and R. A. Bortolussi Epidemic listeriosis, evidence for transmission by food. N. Engl. J. Med. 308: Schuchat, A., K. Deaver, J. D. Wenger, et al Role of foods in sporadic listeriosis. I. Case control study of dietary risk factors. JAMA 27: Schwartz, B., D. Hexter, C. V. Broome, G. R. Brown, A. W. Hightower, R. B. Hirschhorn, J. D. Porter, and P. S. Hayes Investigation in an outbreak of listeriosis: new hypotheses for the etiology of epidemic Listeria monocytogenes. J. Infect. Dis. 9: Spitzer, P. G., S. M. Hammer, and A. W. Karchmer. 98. Treatment of Listeria monocytogenes infection with trimethoprimsulfamethoxazole: case report and review of the literature. Rev. Infect. Dis. 8: Tulkens, P. M. 99. Intracellular pharmacokinetics and localization of antibiotics as predictors of their efficacy against intraphagocytic infection. Scand. J. Infect. Dis. 74(Suppl.): Tulkens, P. M. 99. Intracellular distribution and activity of antibiotic. Eur. J. Clin. Microbiol. Infect. Dis. 0: Van der Auwera, P., T. Matsumoto, and M. Husson Intraphagocytic penetration of antibiotics. J. Antimicrob. Chemother. 22: Winslow, D. L., J. Damme, and E. Dieckman Delayed bactericidal activity of 3-lactam antibiotics against Listeria monocytogenes: antagonism of chloramphenicol and rifampin. Antimicrob. Agents Chemother. 23:-8. Downloaded from on April, 8 by guest