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Antimicrobial Agents and Chemotherapy, August 2003, p. 2545-2550, Vol. 47, No. 8
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.8.2545-2550.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Subinhibitory Concentrations of the Deformylase Inhibitor Actinonin Increase Bacterial Release of Neutrophil-Activating Peptides: a New Approach to Antimicrobial Chemotherapy

Huamei Fu,1* Claes Dahlgren,1 and Johan Bylund2

The Phagocyte Research Laboratory, Department for Rheumatology and Inflammation Research, University of Göteborg, Göteborg, Sweden,1 British Columbia Research Institute for Children's and Women's Health, Vancouver, British Columbia V5Z 4H4, Canada2

Received 2 December 2002/ Returned for modification 10 March 2003/ Accepted 24 May 2003


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ABSTRACT
 
Bacterial protein synthesis starts with a formylated methionine residue, and this residue is sequentially cleaved away by a unique peptide deformylase (PDF) and a methionine aminopeptidase to generate mature proteins. The formylation-deformylation of proteins is a unique hallmark of bacterial metabolism and has recently become an attractive target for the development of antimicrobial agents. The innate immune system uses the formylation of bacterial proteins as a target, and professional phagocytes, e.g., neutrophils, express specific receptors for bacterium-derived formylated peptides. Activation of formyl peptide receptors (FPR) mediates neutrophil migration and the release of oxygen radicals and other antimicrobial substances from these cells. We hypothesize that the use of a PDF inhibitor would increase the production of proinflammatory peptides from the bacteria and thus trigger a more pronounced innate immune response. We tested this hypothesis by exposing Escherichia coli to subinhibitory doses of the PDF inhibitor actinonin and show that actinonin indeed increases the production and secretion of neutrophil-activating peptides that activate human neutrophils through FPR. These findings could be potentially used as a new approach to antibacterial chemotherapy.


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INTRODUCTION
 
Protein synthesis is a process in which bacterial and mammalian cells possess many similarities, but there are enough differences to allow selective blocking of the process in bacteria. Accordingly, the protein synthesis machinery is the target for a wide range of commonly used antimicrobial drugs, such as aminoglycosides, macrolides, tetracycline, and oxazolidinones. Both prokaryotes and eukaryotes initiate protein synthesis with a methionine residue, and this amino acid functions merely as an inducer of the process and has to be removed by an aminopeptidase later in the process in order to generate a mature protein (24, 30). In contrast to mammalian protein synthesis, the initiating methionine residue in prokaryotes (as well as in mitochondria) is formylated by an N-methionyl-tRNA transformylase (2, 23). Further, in order for the bacterial (but not the mitochondrial) aminopeptidase to function properly, it has to work together with a protein/peptide deformylase (PDF). Mature proteins are generated through sequential removal of the N-formyl group and methionine by PDF and the aminopeptidase, respectively (1, 33). Failure to remove the formyl group results in protein synthesis arrest and subsequent inhibition of microbial growth (25). The specific processes of methionine transformylation and deformylation are targets for potential antimicrobial drugs that have not been exploited until recently (36).

The fact that formylated peptides and proteins are specific signatures of bacterial metabolism also makes them attractive targets for our innate immune system, which has adopted a strategy by which to recognize a few highly conserved non-self molecules present on or in a large group of potentially pathogenic microbes (26). Accordingly, the professional phagocytes (granulocytes, monocytes, macrophages) that form our first line of defense against invading microbes (14) express such pattern recognition receptors, for which the N-formylated methionyl group is a critical determinant (35). Because the signals generated by the occupied formyl peptide receptors (FPR) induce chemotaxis, it has been widely held that these receptors evolved to mediate trafficking of phagocytes to sites of bacterial infection. This notion is supported by the fact that N-formyl peptides, in addition to being chemotactic, also possess other proinflammatory properties, such as the ability to activate phagocytes to release antimicrobial peptides and oxidants (30). The importance of proper recognition of formylated peptides is clearly illustrated by the fact that FPR deficiencies are associated with increased susceptibility to bacterial infections (15, 18).

Actinonin is a naturally occurring antibacterial agent (16) that was recently shown to be a potent PDF inhibitor exhibiting antibacterial activity against a wide range of different bacterial species (9). In this study, we have used actinonin to test the hypothesis that bacteria growing in subinhibitory (sublethal) concentrations of a PDF inhibitor release increased amounts of neutrophil-activating peptides with proinflammatory activities. We found that Escherichia coli growing in the presence of actinonin released larger amounts of substances mediating chemotactic migration of neutrophils than did bacteria growing in the absence of the drug. Furthermore, the substances in supernatants from bacteria grown in the presence of actinonin also activated neutrophils to produce larger amounts of superoxide anion than did those from control bacteria. The proinflammatory effects were found to be mediated via the neutrophil FPR, indicating that the neutrophil-activating substances are indeed formylated peptides. These findings indicate that PDF inhibitors, when used in vivo, could have two different effects with implications for bacterial growth, a direct bacteriostatic or bactericidal effect and an indirect proinflammatory effect, which could potentially be used as a new approach to antibacterial chemotherapy.


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MATERIALS AND METHODS
 
Isolation of human neutrophils. Neutrophil granulocytes were isolated from buffy coats obtained from apparently healthy adults. After dextran sedimentation at 1 x g, hypotonic lysis of the remaining erythrocytes, and centrifugation in a Ficoll-Paque gradient (4), the neutrophils were washed twice and resuspended (107/ml) in Krebs-Ringer phosphate buffer containing glucose (10 mM), Ca2+ (1 mM), and Mg2+ (1.5 mM) (KRG; pH 7.3). The cells were stored on melting ice and used within 120 min of preparation.

Cultivation of bacteria. The model bacterial strain used in this study was E. coli MG1655 grown overnight in Luria broth. Bacteria (106 CFU) from such cultures were spread and allowed to grow overnight at 37°C on Luria agar plates with or without different concentrations of actinonin. Plates containing actinonin at a dose that has no effect on bacterial growth (routinely 20 µg/ml for strain MG1655) were used to collect bacterial growth products (GP) as previously described (22). Briefly, the soluble bacterial metabolites were collected by washing the plates with cold KRG. The bacteria were removed by centrifugation and filtration through a filter with a pore size of 0.45 µm. The bacterium-free supernatants were kept on ice until use. The six clinical isolates of E. coli (isolated from patients with urinary tract infections) used in this study were handled as described above, except for the actinonin concentrations used (625 µg/ml for five isolates and 62.5 µg/ml for one isolate).

Neutrophil chemotaxis. Neutrophil chemotaxis was determined with Chemo-Tx multiwell chambers (Neuroprobe Inc., Gaithersburg, Md.) in accordance with the instructions given by the manufacturer. Briefly, neutrophils and the bacterium-free supernatant (corresponding to 5 x 108 CFU/ml) were diluted as indicated in KRG supplemented with bovine serum albumin (0.3%, wt/vol), and 30 µl of the neutrophil suspension (106/ml) was placed on top of a polycarbonate filter with a pore size of 3 µm. The bacterium-free supernatants were applied to the lower compartment (below the filter), the neutrophils were allowed to migrate through the filter, and the accumulation of cells in the lower compartments was determined after 90 min of incubation at 37°C. For quantification, the myeloperoxidase content of the migrated cell lysates was determined by addition of a peroxidase substrate (o-phenylenediamine; Dako A/S, Glostrup, Denmark).

Neutrophil NADPH-oxidase activity. Neutrophil superoxide anion production was determined with an isoluminol-enhanced chemiluminescence (CL) system (12). CL activity was measured in a six-channel Biolumat LB 9505 (Berthold Co., Wildbad, Germany) with disposable 4-ml polypropylene tubes and a 0.5-ml reaction mixture. Tubes containing isoluminol (2 x 10-5 M), horseradish peroxidase (2 U), and neutrophils (106/ml) were allowed to equilibrated for 5 min at 37°C, after which 0.05 ml of GP (diluted in KRG as indicated) was added and the light emission was recorded continuously (details of the CL technique are given in reference 12). In some experiments, the FPR-specific antagonist butoxycarbonyl-Phe-Leu-Phe-Leu-Phe-Leu (Boc-PLPLPL) (final concentration, 10-5 M) or cyclosporine H (final concentration, 10-6 M) was coincubated with the reaction mixture for equilibration before the addition of GP. Lipopolysaccharide (LPS)-primed cells were obtained with LPS from E. coli serotype O111:B4. Cells (107/ml) were incubated in the presence or absence of LPS (10 µg/ml; this relatively high concentration is required to induce priming in the absence of serum) at 37°C for 30 min and then directly used for NADPH-oxidase activation studies.

Chemicals. Horseradish peroxidase was purchased from Boehringer-Mannheim (Mannheim, Germany). The deformylase inhibitor actinonin was obtained from Sigma Chemical Co. (St. Louis, Mo.), as were isoluminol, formylmethionyl-leucyl-phenylalanine (fMLF), LPS, and Boc-PLPLPL. Dextran and Ficoll-Paque were from Pharmacia (Uppsala, Sweden). The hexapeptide Trp-Lys-Tyr-Met-Val-Met (WKYMVM) was synthesized and purified by high-performance liquid chromatography by Alta Bioscience (University of Birmingham, Birmingham, United Kingdom), and cyclosporine H was kindly provided by Novartis Pharma (Basel, Switzerland).


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RESULTS
 
Increased release of neutrophil chemoattractants from E. coli bacteria growing in subinhibitory concentrations of actinonin. In order to investigate the effect of actinonin on bacterial growth, Luria-Bertani plates with serial dilutions of the drug were seeded with approximately 106 CFU/plate and growth was monitored after overnight incubation at 37°C. Complete inhibition of E. coli MG1655 growth was achieved at actinonin concentrations exceeding 80 µg/ml, and the drug had no inhibitory effects at or below 20 µg/ml. Therefore, we continuously used an actinonin concentration of 20 µg/ml for strain MG1655.

The bacterial GP harvested from our control E. coli grown overnight on plates in the absence of any antibiotics (GP-act) contain neutrophil chemoattractants, as illustrated by the ability to attract cells in a chemotaxis filter assay system (Fig. 1). This result is in agreement with data published earlier showing that a number of different bacteria, including E. coli, can produce and release neutrophil chemoattractants during growth (7, 24, 28). The presence of a subinhibitory concentration of the PDF inhibitor actinonin in the bacterial growth medium resulted in increased release of chemoattractants into the bacterium-free supernatant (GP+act). The maximal migration induced by GP+act was comparable to the maximal migration achieved with the positive control fMLF (final concentration, 10-8 M), and the GP+act attracted more cells than GP-act, even when diluted 10 times more (Fig. 1). Actinonin per se had no effect on neutrophil migration (data not shown).



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  		FIG. 1. Subinhibitory doses of actinonin affect the production and release of neutrophil chemoattractants. Transmigration of human neutrophils in response to bacterial GP is shown, where GP+act represents strain MG1655 bacteria (corresponding to 5 x 108 CFU/ml) grown in the presence of actinonin (dark gray) and GP-act represents control bacteria grown in the absence of any antibiotics (light gray). The material was further diluted as indicated. Transmigration in response to fMLF (10-8 M; black) and buffer (background; white) is also shown. Migration levels were measured after 90 min of incubation, and the mean plus the standard deviation of triplicate samples in a representative experiment is shown.

Release of neutrophil NADPH-oxidase-activating peptides is actinonin concentration dependent. Neutrophil NADPH-oxidase-mediated superoxide anion production was measured to quantify neutrophil activation. Neutrophils were exposed to the same density of GP+act obtained from the bacteria grown in the presence of different (subinhibitory) concentrations of actinonin, and NADPH-oxidase activity was monitored. The oxidase activity induced by GP+act increased in an actinonin concentration-dependent manner (Fig. 2). GP-act obtained from bacteria that received no antibiotics induced a very rapid neutrophil response with a time course similar to that induced by well-known chemoattractants such as the formylated peptide fMLF (Fig. 3A). The activity induced (less than 20 x 106 cpm) could not be increased by the use of even more concentrated GP-act (data not shown). GP+act activated the neutrophil NADPH-oxidase with the same response time course but with a maximal level (around 70 x 106 cpm) substantially higher than that achieved with the same density of GP-act (Fig. 3B). In agreement with the results obtained with GP-act, the activity induced by GP+act could not be further increased by the use of a more concentrated solution, but GP+act gave rise to the same oxidative response as GP-act even when diluted eight times (Fig. 3B). Actinonin per se had no effect on neutrophil NADPH-oxidase activity (data not shown). To determine whether this increased secretion of proinflammatory molecules was specifically mediated by inhibition of the bacterial PDF, we exchanged actinonin for subinhibitory doses of kanamycin, which performs its antibiotic effect through interference with the binding of formylmethionyl-tRNA to the ribosome. The CL response induced by GP+kanamycin was similar to that induced by GP-kanamycin. To exclude the possibility that kanamycin might inactivate the CL response induced by GP+kanamycin, kanamycin was added to the cells before fMLF stimulation and we could not detect any inhibitory effect of kanamycin on the fMLF-induced response (data not shown). This indicates that the enhanced production of proinflammatory molecules is specifically mediated by inhibition of bacterial PDF.



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  		FIG. 2. Actinonin increases bacterial production and release of neutrophil NADPH-oxidase activators in a dose-dependent manner. GP+act (corresponding to 5 x 108 CFU/ml) from strain MG1655 grown in the presence of the different subinhibitory concentrations of actinonin indicated was used to activate the neutrophil NADPH-oxidase. The amounts of neutrophil superoxide production (corresponding to the peak value of the response; see Fig. 3) were determined, and the results are presented as the mean ± the standard deviation from four independent experiments. Mcpm, 106 counts per minute.



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  		FIG. 3. Actinonin affects bacterial production and release of neutrophil NADPH-oxidase activators. GP (corresponding to 1.4 x 109 CFU/ml) from strain MG1655 grown in the absence (A) or presence (B) of actinonin (20 µg/ml) were diluted as indicated and used for stimulation of human neutrophils. The resulting superoxide anion production was recorded continuously by isoluminol-enhanced CL, and the results of a representative experiment are shown. Mcpm, 106 counts per minute.

The released bacterial GP activate neutrophils through the formyl peptide receptor. The neutrophil response to a variety of activators (including fMLF) can be primed by LPS through granule mobilization (3, 5). The NADPH-oxidase activity induced by GP+act in LPS-primed neutrophils was two to three times higher than the response in unprimed cells (Fig. 4), suggesting that the E. coli-derived activator in the presence of actinonin uses mobilizable receptors such as FPR or FPR-like receptor FPRL1. The facts that actinonin is a deformylase inhibitor and that the response induced by GP+act resembles that of the formylated peptide fMLF suggest that activation is achieved through occupation of the FPR. To test this hypothesis, we first performed receptor desensitization experiments. Binding of fMLF to its receptor, FPR, results in receptor phosphorylation and subsequent desensitization; i.e., the cells are unable to respond to a new dose of the same agonist. However, they are fully responsive to an agonist that activates the cells through another receptor, e.g., FPRL1 (10). Triggering neutrophils with fMLF not only affected the response to a new dose of fMLF (data not shown) but also desensitized these cells to GP+act (Fig. 5A). Triggering neutrophils with the hexapeptide Trp-Lys-Tyr-Met-Val-Met-NH2 (WKYMVM), a specific FPRL1 agonist, affected the response to a new dose of the same ligand (data not shown), but these cells still responded to GP+act (Fig. 5B). These results suggest that the GP+act released from bacteria are formylated peptides that activate the neutrophils through FPR. To further test this hypothesis, we pretreated neutrophils with the FPR-specific antagonist cyclosporine H; superoxide production in response to GP+act was totally blocked in the presence of cyclosporine H (Fig. 4). A block of GP+act-induced superoxide production was also achieved with another FPR antagonist, Boc-PLPLPL (data not shown).



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  		FIG. 4. Priming and inhibition of the neutrophil NADPH-oxidase response induced by GP+act. Cells were treated with LPS (dotted line), cyclosporine H (broken line), or buffer (solid line) for 30 min at 37°C before stimulation with GP+act (corresponding to 5 x 108 CFU/ml). The resulting superoxide anion production was recorded by isoluminol-enhanced CL, and the results of a representative experiment are shown. Mcpm, 106 counts per minute.



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  		FIG. 5. Desensitization of the neutrophil NADPH-oxidase response induced by GP+act. Neutrophils were either left untreated (solid lines) or pretreated (broken lines) with the FPR agonist fMLF (10-8 M) (A) or the FPRL1 agonist WKYMVM (10-7 M) (B) for 6 min before recording of superoxide anion production was started upon addition of GP+act (corresponding to 5 x 108 CFU/ml). The results of a representative experiment are shown. Mcpm, 106 counts per minute.

Taken together, these results strongly suggest that the proinflammatory molecules released from bacteria growing in the presence of subinhibitory concentrations of actinonin are indeed formylated peptides.

Clinical isolates of E. coli growing in subinhibitory concentrations of actinonin release elevated levels of formylated peptides. To test if actinonin can also cause clinically relevant bacterial strains to produce and release higher levels of neutrophil-activating formylated peptides, six different clinical E. coli isolates from patients with urinary tract infections were investigated. The doses of actinonin required for total inhibition of growth were generally higher for the clinical isolates than for strain MG1655, but as described above for strain MG1655, the optimal subinhibitory concentrations for each of the clinical isolates were titrated and then used for further experiments. The GP+act from all six clinical isolates activated neutrophils to generate and release levels of superoxide anion more than 10-fold higher than the response activated by the corresponding GP-act control supernatants (Table 1). Up to 90% of the neutrophil oxidative responses induced by the GP+act from the clinical isolates were inhibited by the FPR-specific antagonist Boc-PLPLPL (Table 1).


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  		TABLE 1. Subinhibitory concentrations of actinonin enhance the production of neutrophil-activating substances from clinical E. coli isolatesa


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DISCUSSION
 
Currently used antibiotics work by killing or inhibiting the growth of bacteria, which are then removed from the infected tissue by the immune system. Our data showing that an inhibitor of the bacterial enzyme PDF causes increased microbial production and secretion of proinflammatory peptides opens the door for new approaches in the field of antimicrobial chemotherapy. Increased production of chemotactically active peptides, locally at the site of microbial invasion, should increase the recruitment of professional phagocytes, such as neutrophils, to the site of infection. The relative importance of the different parts of the neutrophil killing arsenal in the local defense against a microbial pathogen is not known, but increased recruitment should (per se) allow the innate immune system to more rapidly catch up with and eliminate the invading microbes. The deformylase is essential in bacterial protein synthesis but absent from mammalian cells, making it an attractive target for the development of new drugs to be used for antimicrobial chemotherapy. The facts that several lower eukaryotes, such as malaria parasites and trypanosome species, are equipped with genes homologous to that for PDF and that orthologues of deformylase seem to be present in all bacterial genomes (27) suggest that the phagocyte recruitment strategy could be applied to many different infectious agents and diseases. We could not find any proinflammatory effects of subinhibitory doses of an inhibitor of bacterial protein synthesis (kanamycin), but it has been reported that other antimicrobial agents (e.g., tetracycline) can depress bacterial production of neutrophil-activating peptides (21). Taken together, these findings may potentially be used to strengthen a compromised innate immune response and limit an exaggerated inflammatory response, respectively.

Actinonin, the naturally occurring antimicrobial substance used in this study, was described some 40 years ago, but it was not until recently that it was shown to be a PDF inhibitor (9). PDF catalyzes the removal of the N-formylated group from newly synthesized proteins before methionine aminopeptidase can remove the deformylated methionine. It is thus reasonable to hypothesize that bacteria growing in the presence of subinhibitory concentrations of PDF inhibitors increase their production and release of N-formylated peptides. Our data showing increased release of neutrophil-activating peptides from strain MG1655 cells grown in the presence of actinonin support this hypothesis. The suggestion that the increased level of neutrophil-activating peptides released from bacteria in the presence of actinonin really reflects the presence of formylated peptides is based on the following findings: (i) the kinetics of superoxide production resembles that induced by fMLF; (ii) upregulation of FPR to the cell surface by LPS results in an enhanced (primed) response to fMLF, as well as GP+act; (iii) desensitization of FPR, but not of FPRL1, abolished the GP+act-induced response; and (iv) FPR-specific antagonists inhibit the response.

The deformylation of microbial proteins, catalyzed by PDF, is an essential part of the synthesis machinery for bacteria, as illustrated by the fact that deletion of the pdf gene arrests bacterial growth (8, 33). PDF mutants (which are also resistant to formylase inhibitors) can survive only if the host bacterium also has a mutation in the gene coding for the transformylase. This is the enzyme required to formylate methionine and, as illustrated by the fact that a functional deficiency in this gene alone results in severely impaired bacterial growth, the whole formylation-deformylation process is vital for microbial survival and growth (11, 17). The system is by no means perfect, however, and it has been known for a long time that microbial metabolic activity is associated with a low level of N-formyl peptide release (7, 24, 28). This may be the result of a "leaky" metabolic system, but it has also been suggested that such peptides represent N-terminal signal peptides from newly synthesized bacterial proteins, which are cleaved by prokaryotic signal peptidases present in the bacterial cell wall (24). The efficiency and rate of deformylation are most probably also affected by the sequence of amino acids in the N terminus, as well as their susceptibility to oxidation (31).

It has been known for more than 20 years that formylated peptides are recognized by specific G-protein-linked, seven-transmembrane-spanning receptors on professional phagocytes such as neutrophils and monocytes/macrophages (29). Since N-formyl peptides are products of bacterial metabolism, and their binding to a specific phagocyte receptor (FPR) induces chemotaxis and activation of phagocytes that are critical effector cells in our innate immune system, it is reasonable to assume that the interaction between these two counterparts (formylated peptides and FPR) is important in the antimicrobial host defense. It is interesting that some bacteria, such as Staphylococcus aureus, can secrete molecules that inhibit the formylated-peptide-coupled response in neutrophils (34). Direct proof of the importance of these two counterparts in vivo has been obtained recently with mice lacking the orthologue of human FPR. These animals show increased susceptibility to Listeria infections, clearly illustrating that the functional repertoire induced by N-formyl peptides is of importance for a proper antibacterial host defense (15). It should be noted that the impaired antimicrobial host defense is manifested in FPR-/- animals although the murine orthologue of the human FPR requires 100-fold higher concentrations of N-formylated ligand to become activated (6). Additionally, neutrophils with dysfunctional FPR have been described in patients suffering from localized juvenile periodontitis (18, 32). Functionally intact FPR in human neutrophils is thus of importance for the host defense against Actinomyces actinomycetemcomitans, the microbe responsible for the localized juvenile periodontitis.

PDF belongs to one of the more extensively studied enzyme groups, the metalloproteases, and these enzymes are excellent precedents for a rational approach to drug design. Accordingly, the use of PDF as a potential target for antimicrobial drug design was introduced in the late 1990s (20), and an increasing number of different PDF inhibitors have been constructed and described during the last couple of years. Most of them have the same generic inhibitor structure as actinonin (36), but in contrast to actinonin, some of the newly described PDF inhibitors (11, 13, 19) are also active in vivo when administered both intravenously and orally. More studies are needed for successful development of PDF inhibitors as a new class of chemotherapeutic agents with a dual function as microbial growth inhibitors and amplifiers of the innate immune reaction. Such chemotherapeutic agents could be a valuable complement to the shrinking arsenal of antibiotics available for the treatment of infections caused by an increasing number of multidrug-resistant bacteria.


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ACKNOWLEDGMENTS
 
This work was supported by the Swedish Medical Research Council, the King Gustaf V 80-Year Foundation, the Scandinavian Society for Antimicrobial Chemotherapy, and the Swedish Foundation for Strategic Research—Infection & Vaccinology.

We are grateful to Christine Wennerås for providing the clinical E. coli isolates.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Rheumatology and Inflammation Research, University of Göteborg, Guldhedsgatan 10, 413 46 Göteborg, Sweden. Phone: 46-313424635. Fax: 46-31828898. E-mail: Huamei.Fu{at}microbio.gu.se. Back


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Antimicrobial Agents and Chemotherapy, August 2003, p. 2545-2550, Vol. 47, No. 8
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.8.2545-2550.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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