Effects of trimethoprim and co-trimoxazole on the morphology of Listeria monocytogenes in culture medium and after phagocytosis
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(2) P. Minkowski et al. unstable substances,13 folinic acid (calcium folinate) was used.. Materials and methods Organisms L. monocytogenes strain EGD was originally obtained from B. Blanden, Canberra, Australia. A single colony was subcultured in brain–heart infusion (BHI) broth (Difco Laboratories, Detroit, MI, USA) and stored in aliquots of 2 mL at –70C until use. For experiments, aliquots were diluted 1:46 in BHI broth and grown overnight at 37C to mid-log phase. Bacteria were then washed twice in Gey’s balanced salt solution (GBSS, Gibco Europe, Basel, Switzerland). For experiments with phagocytosis by human blood-derived macrophages, bacteria were opsonized for 15 min at 37C in a mixture of 50% normal human serum and 50% Iscove’s modified Dulbecco’s medium (IMDM, Gibco) and washed twice more before use, as described previously.19 The L. monocytogenes rough mutant RIII was kindly provided by A. Bubert, Lehrstuhl für Mikrobiologie, Biologisches Zentrum, Universität Würzburg, Germany and kept at –70C in BHI broth until use.. MEC, MIC and MBC determinations Two-fold serial dilutions of antibacterial agents were prepared in IMDM supplemented with 10% fetal calf serum (FCS; PAA, Linz, Austria) and 0.13 U/mL thymidine phosphorylase (Sigma Chemie, Buchs, Switzerland) to inactivate any thymidine in the medium. Concentrations of co-trimoxazole, TMP and SMX from 0.01 to 20 mg/L were examined. Concentrations for co-trimoxazole always refer to the concentration of TMP in the combination. Washed overnight cultures of bacteria were diluted in IMDM and inoculated into the serial dilutions of antibiotics to give a final volume of 2 mL in glass tubes (Corning, NY, USA) at a concentration of c. 9 106 cfu/mL. For morphological evaluation of bacteria, glass tubes were incubated at 37C for 6 h. Duplicate samples were prepared from each tube, stained with methylene blue and examined by light microscopy at a magnification of 100 under oil immersion. The minimal concentrations of the antibacterial agents resulting in bacterial elongation (MECs) were recorded by eye. MICs of the antibacterial agents were read after 18 h of incubation at 37C. For the determination of bactericidal activity, MIC tubes showing no growth were appropriately diluted in water and spread on to solid BHI agar plates containing 10 mg/L of thymidine. After incubation for 18 h at 37C, surviving bacteria were determined by counting colonies. MBCs were defined as the lowest concentration of an antimicrobial agent resulting in a 2 log10 reduction of cfu with respect to the starting inoculum. To investigate possible antagonistic effects of folinic acid and pABA on the effect of TMP, serial dilutions of TMP were supplemented with a 15-fold concentration of folinic acid (up to 300 mg/L) or five-fold concentration of pABA (up to 100 mg/L). Since FH2 and FH4 are known to be. Studies of post-antimicrobial effects and reversibility of bacterial elongation For the study of post-antimicrobial effects, cultures previously exposed for 6 h to IMDM containing 0.6 mg/L TMP and 130 U/L thymidine phosphorylase, were washed three times with pre-warmed GBSS after centrifugation at 2000g for 10 min, and incubated in 12 mL of pre-warmed drugfree IMDM with 10% FCS in polypropylene plastic tubes (Falcon, Becton Dickinson, NJ, USA) at 37C in a waterbath on an orbital shaker. Numbers of cfu were determined during the next 6 h and bacterial morphology studied as described above. Control cultures not exposed to TMP were processed in parallel. To quantifiy post-antimicrobial effects on bacterial morphology, cells were counted under the aspect of elongated bacteria, chains and short cells. To determine whether TMP-induced elongation was reversed by addition of p60 protein, concentrated cell-free supernatants containing p60 were prepared from overnight cultures of L. monocytogenes strain EGD as described previously,20 and added to elongating concentrations of TMP.. Morphological studies of intracellular L. monocytogenes exposed to co-trimoxazole and TMP after phagocytosis by murine J774 and human blood-derived macrophages The murine macrophage cell line J774 (ATCC no. HB-197) was cultured in IMDM supplemented with 10% FCS (complete medium) at 37C, 5% CO2 and 98% humidity. For experiments, J774 cells were seeded on to 13 mm diameter plastic coverslips (Thermanox, Nunc, Naperville, IL, USA) in 24-well tissue culture plates (Falcon). Macrophages were allowed to adhere for 1 h before a challenge with c. 1.8 107 bacteria per coverslip. After 15 min of incubation for phagocytosis, non-phagocytosed bacteria were removed by three washes with pre-warmed GBSS. Complete medium, containing the same range of two-fold serial dilutions of cotrimoxazole or TMP and supplemented with thymidine phosphorylase as described for the extracellular experiments, was added to a final volume of 1 mL. Coverslips were removed after 6 h of incubation and bacterial morphology analysed after Giemsa staining. Bacterial length was measured by light microscopy with an axioscope under 100 oil immersion. Human peripheral blood mononuclear cells were isolated from heparinized blood of normal volunteers by Ficoll–Hypaque density centrifugation (Pharmacia, Uppsala, Sweden) as described previously.21 Cells suspended in IMDM supplemented with 20% pooled human serum (complete medium) were seeded on to coverslips. 186.
(3) Morphological effects of trimethoprim on Listeria at a density of 6 106 cells/coverslip. After adhesion for 2 h at 37C, 5% CO2 and 98% humidity, non-adherent cells were removed by three washes with GBSS. To render human blood-derived macrophages receptive for intracellular listerial growth, cells were incubated for 48 h in complete medium with the following deactivating agents: 2 g/L IL-4, 5 g/L IL-10 or 2.5 10–7 M dexamethasone.22 Medium and deactivating agents were changed after 24 h during the 48 h deactivation period. Subsequently, human blood-derived macrophages were challenged with 1.8 107 opsonized bacteria per coverslip as described above. Medium and deactivating agents were replaced and different concentrations of TMP (0.16, 0.31, 0.63, 1.25 and 5 mg/L) added to control and deactivated cell cultures. Coverslips were removed after 6 h of incubation and analysed after Giemsa staining as already described. Bacterial length was again measured by light microscopy with an axioscope under 100 oil immersion. Thymidine was not removed by thymidine phosphorylase in these experiments.. Electron microscopy For electron microscopy, bacterial suspensions or cells on plastic coverslips were fixed in 2.5% gluteraldehyde plus 0.8% paraformaldehyde (both in 0.05 M cacodylate buffer) and processed using standard methods.22. Reagents, antibacterials and cytokines Co-trimoxazole (sulphamethoxazole:trimethoprim 5:1; Roche Pharma, Reinach, Switzerland) and the calcium salt of 5-formylfolic acid (calcium folinate; Farmos, Baar, Switzerland) were obtained as solutions. SMX (Roche Pharma) and pABA (Sigma) were dissolved in 100% ethanol. Thymidine (Sigma) was dissolved in distilled water and TMP (Sigma) in distilled water with 0.1% dimethylsulphoxide (Sigma). Dexamethasone (Sigma) was dissolved in 100% ethanol. Human recombinant IL-4 (Becton Dickinson) had a specific activity of 2 106 U/mg and human recombinant IL-10 (Pepro-Tech, Rocky Hill, NJ,. USA) an ED50 of 2 g/L in an MC-90 costimulation assay, as specified by the manufacturers. Solvents were tested on control cultures and shown to have no effect on the parameters examined at the concentrations used. Each drug was of known potency and freshly prepared before use.. Statistical analyses Values are given as mean standard deviation (S.D.) as indicated. When measuring bacterial length, differences between control cultures and treated bacteria were evaluated for statistical significance by one-way ANOVA for unpaired data with more than two groups, and by Dunn’s Multiple Comparison post test (Graphpad Prism; Graphpad Software, San Diego, CA, USA).. Results Effects of co-trimoxazole, TMP and SMX on the morphology and in vitro susceptibility of extracellular L. monocytogenes In pilot experiments we observed that L. monocytogenes elongated several fold in the presence of co-trimoxazole (Figure 1a versus b), an effect well known in Gram-positive cocci treated with β-lactam antibiotics that are active against cell walls.23,24 As judged by light microscopy, exposure for a few hours to co-trimoxazole or TMP alone, but not to SMX, induced Listeria to elongate at very low concentrations (Table). Single elongated bacteria, as well as several bacteria appearing unable to separate from each other, were observed, as demonstrated for one TMP concentration resulting in pronounced elongation (Figure 1a versus b). Electron microscopy confirmed listerial elongation in response to co-trimoxazole and TMP (Figure 2). Two effects could clearly be distinguished: elongation occurred either in the form of single elongated bacteria, sometimes with abortive septation (Figure 2b and e), or in the form of several bacteria remaining attached to each other, forming a chain (Figure 2c and d). At high concentrations of co-trimoxazole and TMP, lysis of cell matrix,. Table. MIC, MBC and MEC values (mg/L) of co-trimoxazole, TMP and SMX for extracellular L. monocytogenesa MIC Test agent Co-trimoxazole TMP SMX. MBC. MEC. mean. range. mean. range. mean. range. 0.05 0.83 20. 0.04–0.08 0.63–1.25 20. 0.2 5 20. 0.16–0.31 5 20. 0.02 0.21 20. 0.02 0.16–0.31 20. a. Data are mean and range from three independent experiments.. 187.
(4) P. Minkowski et al.. Figure 1. Photomicrographs of L. monocytogenes strain EGD in cell culture medium (a, b) and after phagocytosis by murine J774 (c, d) or human blood-derived (e, f) macrophages, showing the effects of co-trimoxazole and TMP on the morphology of bacteria. (a, b) Methylene-stained Listeria exposed for 6 h to medium containing 0.16 mg/L co-trimoxazole (a) compared with control culture not exposed to the antibiotic (b). (c, d) Giemsa-stained J774 macrophages 6 h after challenge with bacteria in the presence of medium containing 0.31 mg/L trimethoprim, harbouring elongated Listeria (a), and in drug-free medium (b). (e, f) Giemsa-stained human blood-derived macrophages deactivated for 48 h with IL-4 (e) or IL-10 (f) before challenge with bacteria. Intracellular elongated Listeria exposed for 6 h to medium containing 0.31 mg/L trimethoprim (e) compared with intracellular control bacteria in drug-free medium (f). Bar, 10 m.. membrane disruption, and fragmentation of bacteria were seen (Figure 2d). MECs were determined and compared with their respective MICs and MBCs (Table). Elongation was observed with co-trimoxazole and TMP at a concentration well below the MIC. The effect of SMX in combination with TMP was always synergic with regard to MEC, MIC and MBC, while SMX alone did not affect the morphology. of Listeria (data not shown) and did not restrict bacterial growth (Table). Potential antagonistic effects of folinic acid and pABA on TMP were investigated for extracellular L. monocytogenes. The addition of exogenous calcium folinate or pABA at a 15- or five-fold concentration, respectively, of TMP did not result in an antagonism of TMP with regard to either MEC, MIC or MBC (data not shown).. 188.
(5) Morphological effects of trimethoprim on Listeria. Figure 2. Ultrathin sections of L. monocytogenes strain EGD in cell culture medium (a–d) and after phagocytosis by J774 macrophages (e, f), showing the effects of co-trimoxazole and TMP on the morphology of bacteria. (a) Control culture not exposed to antibiotics. (b) Culture treated with co-trimoxazole (0.16 mg/L): elongated forms of bacteria are clearly visible. (c) Culture treated with trimethoprim (0.63 mg/L): elongated bacteria with abortive septation. (d) Culture treated with co-trimoxazole (0.31 mg/L): chain formation of L. monocytogenes, note cytoplasmic fragmentation and membrane disturption of one bacterium. (e, f) L. monocytogenes inside J774 cells in the presence of co-trimoxazole (0.16 mg/L). Elongated bacteria with incomplete septation (e) and bacteria forming chains (f) are found free in the cytoplasm of the macrophage. Note the well-defined actin coat (arrows) closly attached to the bacterial surface. Bar, 0.5 m.. Reversibility of TMP-induced elongation of L. monocytogenes In subsequent studies we investigated the reversibility of bacterial elongation after removal of TMP. To this end,. L. monocytogenes was exposed for 6 h to an elongating concentration of TMP (0.6 mg/L; Table) before transfer into drug-free medium. Figure 3a shows the bacterial growth rate determined by quantitative cultures, in comparison with control bacteria never exposed to the anti-. 189.
(6) P. Minkowski et al. bacterial agent. TMP caused a marked reduction in cfu during the 6 h exposure time. However, after transfer into drug-free medium the growth rate of bacteria was above the rate of control cultures, with a generation time of approximately 50% of that of control bacteria (Figure 3a). Morphological quantifications showed that after removal. of an elongating concentration of TMP, single elongated bacteria and cell chains became less frequent, with a rapid increase in the number of dividing normal ones. Figure 3b shows the percentage of the total bacteria counted. In parallel, morphological evaluations by light microscopy showed that cell chains and elongated bacteria clearly became shorter at each point in time throughout their growth curve (data not shown). A rough mutant of L. monocytogenes, which is impaired in the synthesis of a major extracellular protein, named p60 for its relative molecular mass of 60 kDa, also forms cell chains.20,25 This effect is reversed after addition of partially purified p60 from the wild-type strain.20 We therefore investigated whether TMP-induced elongation was similarly inhibited by p60. However, addition of p60-containing supernatants to TMP-treated L. monocytogenes strain EGD did not antagonize elongation, in contrast to parallel control experiments with the rough mutant RIII (data not shown).. Morphological effects of TMP and co-trimoxazole on intracellular L. monocytogenes. Figure 3. Post-antimicrobial effects of TMP on L. monocytogenes strain EGD. Bacteria were cultured in the presence of TMP (0.6 mg/L) for 6 h, followed by 6 h in drug-free medium. Control cultures not exposed to TMP were processed in parallel. (a) Growth curves of TMP-exposed () and control () bacteria. Results of viable counts at each point in time are mean value S.D. from triplicate samples for one representative out of two similar experiments. (b) Quantification of different morphological forms of bacteria after removal of TMP. Results at each point in time were obtained by microscopically scoring 200 randomly chosen bacteria from each of triplicate methylenestained preparations under the aspect of elongated bacteria (), chains () and short cells (). Results represent mean values S.D. for one representative of two similar experiments.. Similar to extracellular bacteria, L. monocytogenes replicating inside murine J774 macrophages elongated in response to co-trimoxazole and TMP (Figure 1c and d). The two remarkable effects of single elongated bacteria and bacterial chains could be clearly distinguished again (Figure 2e and f). As expected,26 J774 macrophages were unable to restrict bacterial growth, even in the presence of co-trimoxazole or TMP (data not shown). It is known that L. monocytogenes mobilizes and polymerizes host-cell F-actin, forming a comet-like tail, which enables it to spread from cell to cell.26 Actin coats were still observed on intracellular single elongated Listeria (Figure 2e) and bacterial chains (Figure 2f). Intracellular MECs of co-trimoxazole and TMP and the range of concentrations resulting in elongation were the same as found for extracellular bacteria (data not shown). Intracellular bacterial length was quantified for cultures showing impressive elongation. Most bacteria elongated in the presence of co-trimoxazole (Figure 4a) and TMP (Figure 4b), whereby more than one-third of bacteria was double or triple the length of control bacteria. The mean length of bacteria in the presence of 0.04 or 0.16 mg/L of cotrimoxazole was 2.8 and 3.0 m, and in the presence of 0.31 or 0.63 mg/L TMP it was 2.6 and 3.6 m, respectively. In comparison, unexposed control bacteria were on average 1.2 m long. Addition of 0.63 mg/L TMP caused a greater increase than addition of the lower concentration of TMP (Figure 4b). The overall longest bacterium was measured in a culture exposed to TMP and reached a length of 10.8 m (Figure 4b). The experiments were extended to studies on interactions between TMP and Listeria phagocytosed by human blood-derived macrophages, which were rendered recep-. 190.
(7) Morphological effects of trimethoprim on Listeria tive to intracellular listerial growth by deactivation. Evaluated by light microscopy and consistent with earlier studies in this laboratory,22 deactivation with IL-4 resulted in the highest increase in bacterial numbers, followed by deactivation with IL-10 and then dexamethasone (data not shown). Morphological evaluation proved that Listeria also elongated inside human blood-derived macrophages after exposure to TMP, but not in its absence (Figure 1e and f). Bacterial elongation was quantified for one of the concentrations of TMP tested that induced pronounced elongation (Figure 4c). Again, more than one-third of the bacteria reached double or even triple the length of control cultures. Which deactivating agent was chosen to render human blood-derived macrophages permissive for the growth of Listeria made no difference to the bacteria’s size. Bacteria elongated comparatively within all deactivated phagocytes. To better account for the availability of thymidine in the in vivo situation, media were not rendered thymidine-free in these experiments. The potential availibility of thymidine in experiments with human macrophages had no effect on bacterial morphology in comparison with experiments with murine J774 macrophages in thymidine-free culture media (data not shown).. Discussion. Figure 4. Effects of co-trimoxazole and TMP on the length of intracellular L. monocytogenes strain EGD after phagocytosis by murine (a, b) or human (c) macrophages. (a, b) J774 murine macrophages were challenged with bacteria and cultivated for 6 h in the presence of (a) two different concentrations of cotrimoxazole (, 0.04–0.2 mg/L; , 0.16–0.8 mg/L; , control) or (b) TMP (, 0.31 mg/L; , 0.63 mg/L; , control). Control cultures were challenged in parallel in the absence of antibacterials. Results were determined by measuring 50 randomly chosen bacteria from triplicate samples for one representative out of two similar experiments. For statistical comparison of bacterial elongation, co-trimoxazole- and TMP-exposed bacteria versus control bacteria, all P values were 0.001. Comparison of antibiotic concentrations: 0.16 mg/L of co-trimoxazole versus lower concentration, P 0.05; and 0.63 mg/L TMP versus lower concentration, P 0.05. (c) Human blood-derived macrophages were rendered receptive to intracellular bacterial growth by deactivation with IL-4 (), IL-10 ( ) or dexamethasone (Dex) ( ) for 48 h. Macrophages were challenged with bacteria and cultivated for 6 h in the presence of 0.31 mg/L TMP. Control cultures were challenged in parallel in the absence of TMP (). Results were determined by measuring 50 randomly chosen bacteria from triplicate samples for one representative out of two similar experiments. For statistical comparison of elongation, TMP-exposed bacteria versus control bacteria, all P values were 0.001. Comparison of deactivating agents: IL-4 versus IL-10, IL-4 versus Dex and IL-10 versus Dex, all P values 0.05.. In order to appreciate the activity of antibacterial agents against intracellular bacteria, their intracellular activity has to be taken into account. L. monocytogenes is a facultative intracellular pathogen that can cause severe food-borne infections in humans and animals.27 TMP is used in combination with SMX in the treatment of clinical listeriosis. The present studies report a novel effect of TMP on the morphology of L. monocytogenes, resulting in elongation of bacteria (Figure 1). TMP induced elongation of extracellular bacteria and, more importantly, intracellular bacteria phagocytosed by permissive macrophages. Bacterial elongation occurred even at subinhibitory concentrations of TMP and was observed in two distinct forms: (i) bacteria transformed either into single elongated cells, sometimes with abortive septation; or (ii) occurred in the form of chains with a failure of bacteria to separate (Figures 1 and 2). Despite previous resports that TMP is concentrated in phagocytes,12 we found no increased activity of TMP on the morphology of phagocytosed bacteria. Thus, it remains to be determined whether accumulation of TMP as described previously12 occurs in the same subcellular compartments as accessed by the bacteria. On the other hand, our preliminary observations gave no indication that extra- or intracelllular activity of TMP was affected by the potential availability of thymidine, preformed exogenous folates or pABA, which may be supplied by the intracellular milieu and circumvent its activity15–17 (data not shown). Studies on the post-antimicrobial effects of TMP revealed a percentage reduction of elongated bacteria after. 191.
(8) P. Minkowski et al. removal of TMP (Figure 3b). In parallel, an increased growth rate exceeding that of control cultures resulted (Figure 3a). These phenomena could be explained by a reversal of elongation owing to completion of cell division and separation of elongated cells. However, it cannot be ruled out that subsequent normal sized cells also derived from surviving normal bacteria instead of reverting from the elongated form. To our knowledge, this inhibitory effect of TMP on cell separation of L. monocytogenes has not been described previously, despite its use and importance in clinical therapy. However, early reports on antifolates describe the formation of filamentous cells of Escherichia coli and Aerobacter aerogenes.28 Biochemical studies on the effects of TMP on Enterobacter cloacae showed an effect on cell wall precursors.29 In addition, the Gram-positive Enterococcus faecalis showed alterations in cell wall formation when exposed to TMP,30 and disturbed peptidoglycan synthesis was suggested to be responsible. Further studies are necessary to confirm the disturbance of peptidoglycan synthesis by antifolates such as TMP. The L. monocytogenes rough mutant RIII is known to form cell chains because of a lack of p60 production,20 a suggested murein hydrolase required for a late step in cell division.25 However, our experiments did not indicate any inhibitory effect of TMP on p60 production. The existence of elongation mutants of Listeria in clinical and food samples with normal p60 production has also been reported by others.31 Furthermore, it is of note that we did not observe any obvious effect of TMP on bacterial cell functions. Escape into the cytoplasm, polymerization of actin and formation of so-called comet tails,26 all functions related to the virulence of L. monocytogenes, were not affected (Figure 2e and f). In conclusion, our studies show that TMP, although reported to be concentrated intracellularly,12 did not have an increased intracellular activity. TMP had an effect on cell separation of L. monocytogenes at subinhibitory concentrations without affecting important virulence mechanisms used by the bacterium to escape phagosomes and to access other cells. Finally, altered cell morphology of L. monocytogenes at very low concentrations of TMP might shed light on diagnostic confusion in the morphological evaluation of clinical material as well as primary cultures containing low concentrations of TMP, as elongated rods may be L. monocytogenes.. References. Acknowledgements. 16. Beck, C. F., Ingraham, J. L., Neuhard, J. & Thomassen, E. (1972). Metabolism of pyrimidines and pyrimidine nucleosides by Salmonella typhimurium. Journal of Bacteriology 110, 219–28.. We thank PD Dr G. Schoedon for critical reading of the manuscript and Mrs M. Erni, Mrs S. Märki, Mr L. Hirt and Mr H. Sonderegger for technical help. This work was supported by grant no. 31-457776 from the Swiss National Science Foundation.. 1. Hitchings, G. H. & Burchall, J. J. (1965). Inhibition of folate biosynthesis and function as a basis for chemotherapy. Advances in Enzymology 27, 417–68. 2. Fan, H., Brunham, R. C. & McClarty, G. (1992). Acquisition and synthesis of folates by obligate intracellular bacteria of the genus Chlamydia. Journal of Clinical Investigations 90, 1803–11. 3. Merle-Melet, M., Dossou-Gbete, L., Maurer, P., Meyer, P., Lozniewski, A., Kuntzburger, O. et al. (1996). Is amoxicillin– cotrimoxazole the most appropriate antibiotic regimen for Listeria meningoencephalitis? Review of 22 cases and the literature. Journal of Infection 33, 79–85. 4. Jones, E. M. & MacGowan, A. P. (1995). Antimicrobial chemotherapy of human infection due to Listeria monocytogenes. European Journal of Clinical Microbiology and Infectious Diseases 14, 165–75. 5. Scheld, W. M., Fletcher, D. D., Fink, F. N. & Sande, M. A. (1979). Response to therapy in an experimental rabbit model of meningitis due to Listeria monocytogenes. Journal of Infectious Diseases 140, 287–94. 6. Spitzer, P. G., Hammer, S. M. & Karchmer, A. W. (1986). Treatment of Listeria monocytogenes infection with trimethoprim– sulfamethoxazole: case report and review of the literature. Reviews in Infectious Diseases 8, 427–30. 7. Hof, H., Nichterlein, T. & Kretschmar, M. (1997). Management of listeriosis. Clinical Microbiological Reviews 10, 345–57. 8. Marget, W. & Seeliger, H. P. (1988). Listeria monocytogenes infections—therapeutic possibilities and problems. Infection 16, Suppl. 2, S175–7. 9. Louria, D. B., Hensle, T., Armstrong, D., Collins, H. S., Blevins, A., Krugman, D. et al. (1967). Listeriosis complicating malignant disease. A new association. Annals of Internal Medicine 67, 260–81. 10. Hof, H. (1991). Intracellular microorganisms: a particular problem for chemotherapy. Infection 19, Suppl. 4, S193–4. 11. Michelet, C., Avril, J. L., Cartier, F. & Berche, P. (1994). Inhibition of intracellular growth of Listeria monocytogenes by antibiotics. Antimicrobial Agents and Chemotherapy 38, 438–46. 12. Hand, W. L. & King-Thompson, N. L. (1989). The entry of antibiotics into human monocytes. Journal of Antimicrobial Chemotherapy 23, 681–9. 13. Hamilton-Miller, J. M. (1988). Reversal of activity of trimethoprim against Gram-positive cocci by thymidine, thymine and ‘folates’. Journal of Antimicrobial Chemotherapy 22, 35–9. 14. Jones, C., Stevens, D. L. & Ojo, O. (1987). Effect of minimal amounts of thymidine on activity of trimethoprim–sulfamethoxazole against Staphylococcus epidermidis. Antimicrobial Agents and Chemotherapy 31, 144–7. 15. Okazaki, R. & Kornberg, A. (1964). Deoxythymidine kinase of Escherichia coli. II. Kinetics and feedback control. Journal of Biological Chemistry 239, 275–84.. 17. Carlson, C. A., Stewart, G. J. & Ingraham, J. L. (1985). Thymidine salvage in Pseudomonas stutzeri and Pseudomonas aeruginosa provided by heterologous expression of Escherichia coli thymidine kinase gene. Journal of Bacteriology 163, 291–5.. 192.
(9) Morphological effects of trimethoprim on Listeria 18. Indiveri, M. C. & Hirsh, D. C. (1992). Tissues and exudates contain sufficient thymidine for growth of anaerobic bacteria in the presence of inhibitory levels of trimethoprim–sulfamethoxazole. Veterinary Microbiology 31, 235–42.. 25. Wuenscher, M. D., Kohler, S., Bubert, A., Gerike, U. & Goebel, W. (1993). The iap gene of Listeria monocytogenes is essential for cell viability, and its gene product, p60, has bacteriolytic activity. Journal of Bacteriology 175, 3491–501.. 19. Schaffner, A. & Rellstab, P. (1988). Gamma-interferon restores listericidal activity and concurrently enhances release of reactive oxygen metabolites in dexamethasone-treated human monocytes. Journal of Clinical Investigations 82, 913–9.. 26. Campbell, P. A. (1994). Macrophage–Listeria interactions. Immunology Series 60, 313–28.. 20. Kuhn, M. & Goebel, W. (1989). Identification of an extracellular protein of Listeria monocytogenes possibly involved in intracellular uptake by mammalian cells. Infection and Immunity 57, 55–61. 21. Schaffner, A. (1985). Therapeutic concentrations of glucocorticoids suppress the antimicrobial activity of human macrophages without impairing their responsiveness to gamma interferon. Journal of Clinical Investigations 76, 1755–64. 22. Blauer, F., Groscurth, P., Schneemann, M., Schoedon, G. & Schaffner, A. (1995). Modulation of the antilisterial activity of human blood-derived macrophages by activating and deactivating cytokines. Journal of Interferon and Cytokine Research 15, 105–14. 23. Lorian, V. & Gemmel, C. (1991). Effect of low antibiotic concentrations on bacteria: effects on ultrastructure, virulence, and susceptibility to immunodefenses. In Antibiotics in Laboratory Medicine, 3rd edn, (Lorian, V., Ed.), pp. 493–555. Williams & Willkins, Baltimore, MD. 24. Lorian, V. (1975). Some effects of subinhibitory concentrations of antibiotics on bacteria. Bulletin of the New York Academy of Medicine 51, 1046–55.. 27. Farber, J. M. & Peterkin, P. I. (1991). Listeria monocytogenes, a food-borne pathogen. Microbiology Reviews 55, 476–511. 28. Nickerson, W. J. & Webb, M. (1956). Effect of folic acid analogues on growth and cell division of nonexacting organisms. Journal of Bacteriology 71, 129–39. 29. Richards, E. M. & Xing, D. K. (1994). Separation and quantification of murein and precursors from Enterobacter cloacae after treatment with trimethoprim and sulphadiazine. Journal of Pharmacy and Pharmacology 46, 690–6. 30. Richards, R. M., Xing, J. Z., Gregory, D. W. & Marshall, D. (1995). Mechanism of sulphadiazine enhancement of trimethoprim activity against sulphadiazine-resistant Enterococcus faecalis. Journal of Antimicrobial Chemotherapy 36, 607–18. 31. Rowan, N. J., Dandlish, A. A., Bubert, A., Anderson, J. G., Kramer, K. & McLauchlin, J. (2000). Virulent rough filaments of Listeria monocytogenes from clinical and food samples secreting wild-type levels of cell-free p60. Journal of Clinical Microbiology 38, 2643–8. Received 22 September 2000; returned 26 February 2001; revised 9 April 2001; accepted 17 April 2001. 193.
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