a Research Unit, Medical Clinic B, University Hospital Zurich, Raemistrasse 100, CH-8091 Zurich; b Institute of Anatomy, University of Zurich, CH-8091 Zurich, Switzerland
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Abstract |
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Introduction |
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Co-trimoxazole is used for a variety of bacterial infections, including infections with the facultative intracellular pathogen Listeria monocytogenes, particularly in patients intolerant to ß-lactam antibiotics.3,4 In meningitis caused by L. monocytogenes it has been recommended as first-line treatment because of the good diffusion of SMX and TMP into the meninges.5,6 Listeria spp. cause severe infections such as septicaemia and meningitis, primarily in immunocompromised hosts, with a mortality of up to 30%. This underlines the importance of appropriate antibiotic therapy.7 Therapeutic failure and relapse are described in the literature, including therapy with co-trimoxazole.710 In earlier studies of antibiotic activity on intracellular growth of L. monocytogenes, co-trimoxazole showed only poor, non-dose-dependent, slow bactericidal activity.11
TMP has been shown to be concentrated in phagocytes;12 however, it is not known in which subcellular compartment this accumulation takes place. Several Gram-positive bacteria are able to take up and use exogenous thymidine, circumventing TMP activity.13,14 This phenomenon, or salvage pathway, has been described for various bacteria.1517 In vivo, tissues and exudates may contain sufficient thymidine to antagonize the effects of TMP against bacteria,18 but the accessibility of thymidine for bacteria after phagocytosis has not been studied.
We therefore studied the effects of TMP, SMX and co-trimoxazole on L. monocytogenes after phagocytosis by murine J774 and human blood-derived macrophages as well as in culture medium. The studies revealed a novel effect of TMP on the morphology of L. monocytogenes at very low concentrations, resulting in considerably elongated bacteria. A further aim of this work was to examine possible antagonistic effects of preformed folates and pABA on TMP against extracellular Listeria spp. Such reversal effects on the bacterial susceptiblity are known for folinic acid, FH2 and FH4 against Gram-positive species, such as the group D streptococci treated with TMP.13
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Materials and methods |
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L. monocytogenes strain EGD was originally obtained from B. Blanden, Canberra, Australia. A single colony was subcultured in brainheart infusion (BHI) broth (Difco Laboratories, Detroit, MI, USA) and stored in aliquots of 2 mL at 70°C until use. For experiments, aliquots were diluted 1:46 in BHI broth and grown overnight at 37°C 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 37°C 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 70°C 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 x 106 cfu/mL. For morphological evaluation of bacteria, glass tubes were incubated at 37°C for 6 h. Duplicate samples were prepared from each tube, stained with methylene blue and examined by light microscopy at a magnification of x100 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 37°C. 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 37°C, 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 unstable substances,13 folinic acid (calcium folinate) was used.
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 drug-free IMDM with 10% FCS in polypropylene plastic tubes (Falcon, Becton Dickinson, NJ, USA) at 37°C 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 37°C, 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 x 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 co-trimoxazole or TMP and supplemented with thymidine phosphorylase as described for the extracellular experiments, was added to a final volume of 1 mL. Cover- slips 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 x100 oil immersion.
Human peripheral blood mononuclear cells were isolated from heparinized blood of normal volunteers by FicollHypaque 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 at a density of 6 x 106 cells/coverslip. After adhesion for 2 h at 37°C, 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 µg/L IL-4, 5 µg/L IL-10 or 2.5 x 107 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 x 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 x100 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 x 106 U/mg and human recombinant IL-10 (Pepro-Tech, Rocky Hill, NJ, USA) an ED50 of 2 µg/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).
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Results |
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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 ß-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, membrane disruption, and fragmentation of bacteria were seen (Figure 2d
).
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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).
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 antibacterial 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).
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Morphological effects of TMP and co-trimoxazole on intracellular L. monocytogenes
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 co-trimoxazole was 2.8 and 3.0 µm, and in the presence of 0.31 or 0.63 mg/L TMP it was 2.6 and 3.6 µm, respectively. In comparison, unexposed control bacteria were on average 1.2 µm 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 µm (Figure 4b
).
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Discussion |
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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 activity1517 (data not shown).
Studies on the post-antimicrobial effects of TMP revealed a percentage reduction of elongated bacteria after 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.
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Acknowledgments |
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Notes |
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References |
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Received 22 September 2000; returned 26 February 2001; revised 9 April 2001; accepted 17 April 2001