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Antimicrobial Agents and Chemotherapy, August 2005, p. 3396-3403, Vol. 49, No. 8
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.8.3396-3403.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Azithromycin Inhibits the Formation of Flagellar Filaments without Suppressing Flagellin Synthesis in Salmonella enterica Serovar Typhimurium

Hidenori Matsui,^1,2* Masahiro Eguchi,^1,2 Katsufumi Ohsumi,² Akio Nakamura,³ Yasunori Isshiki,^2, Kachiko Sekiya,⁴ Yuji Kikuchi,^1,2 Tohru Nagamitsu,⁴ Rokuro Masuma,^1,2 Toshiaki Sunazuka,^1,2 and Satoshi Omura^1,2,4

Kitasato Institute for Life Sciences,¹ School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641,⁴ Center for Basic Research, The Kitasato Institute, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8642,² Department of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan³

Received 24 September 2004/ Returned for modification 29 November 2004/ Accepted 29 May 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study shows that a sub-MIC of the macrolide antibiotic azithromycin (AZM) diminishes the virulence function of Salmonella enterica serovar Typhimurium. We first constructed an AZM-resistant strain (MS248) by introducing ermBC, an erythromycin ribosome methylase gene, into serovar Typhimurium. The MIC of AZM for MS248 exceeded 100 µg/ml. Second, we managed to determine the efficacy with which a sub-MIC of AZM reduced the virulence of MS248 in vitro. On the one hand, AZM (10 µg/ml) in the culture medium was unable to inhibit the total protein synthesis, growth rate, or survival within macrophages of MS248. On the other hand, AZM (10 µg/ml) reduced MS248's swarming and swimming motilities in addition to its invasive activity in Henle-407 cells. Electron micrographs revealed no flagellar filaments on the surface of MS248 after overnight growth in L broth supplemented with AZM (10 µg/ml). However, immunoblotting analysis showed that flagellin (FliC) was fully synthesized within the bacterial cells in the presence of AZM (10 µg/ml). In contrast, the same concentration of AZM reduced the export of FliC to the culture medium. These results indicate that a sub-MIC of AZM was able to affect the formation of flagellar filaments, specifically by reducing the amount of flagellin exported from bacterial cells, but it was not involved in suppressing the synthesis of flagellin. Unfortunately, AZM treatment was ineffective against murine salmonellosis caused by MS248.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Salmonella enterica serovar Typhimurium is known as the cause of a self-limiting gastroenteritis in humans but of a typhoid-like systemic infection in mice (9). In a mouse model of oral infection, serovar Typhimurium can invade the intestinal epithelium and reach the deeper tissues of the liver and spleen (14). S. enterica is a facultative intracellular parasite capable of surviving within various types of eukaryotic cells, including phagocytic and epithelial cells (4, 43). The intracellular location of some microorganisms allows them to resist antibiotics such as the ß-lactam compounds (35). In fact, S. enterica is intrinsically resistant to benzylpenicillin, oxacillin, most macrolides, rifampin, lincosamides, streptogramins, glycopeptides, and fusidic acid (46). Chloramphenicol (CHL) has been used as a highly effective antibiotic against S. enterica for many years (12).

The azalide class of antibiotics has lately provided another option for the treatment of salmonellosis (51). Azithromycin (AZM), a new 15-member macrolide antibiotic that is the only azalide antibiotic currently available (51), possesses good in vitro activity against a large number of bacterial enteric pathogens, including S. enterica (10, 12, 42, 46). As a general rule, macrolide antibiotics exert their bacteriostatic or bactericidal action by blocking the protein biosynthetic steps that occur on the 50S subunit of the bacterial ribosome (52). Although AZM was derived from a 14-member erythromycin (ERY) that is a natural polyketide product of the streptomycete Saccharopolyspora erythraea, AZM was generally more effective than ERY against strains of invasive enteric pathogens by virtue of its high and prolonged cellular and tissue concentrations (42). Whereas many antibiotics are active in vitro, they are often inactive in vivo against intracellular bacteria because of their poor penetration of cells or because they are impaired under intracellular conditions (41). Consequently, to be effective, antibiotics must penetrate the cellular milieu. The MIC of AZM for serovar Typhi was reduced in broth exposed to previous growth of serovar Typhi, suggesting that AZM could be more active in tissues than standard MIC measurements have indicated (5, 6). Recently, X-ray examination of the 50S subunit of ERY-soaked ribosome revealed the antibiotic bound in the peptidyltransferase cavity, in the vicinity of both A loops and P loops and near A₂₀₅₈ in the 23S rRNA (53). It is the monomethylation or dimethylation of this N⁶ exocyclic amino group of A₂₀₅₈ by an ERY ribosome methylation (Erm) modification enzyme that produces the Erm phenotype and reduces the RNA's affinity for the antibiotic without affecting the role of A₂₀₅₈ in peptidyltransferase architecture or function (53).

Despite the considerable efficacy of AZM, little information is available regarding the actions of antibacterial mechanisms against Salmonella infection. Under the assumption that macrolide antibiotics promote both the deterioration of virulence factors and the inhibition of bacterial protein synthesis, we have been studying the bacteriostatic efficacy of macrolide antibiotics using in vivo and in vitro models of Salmonella infections. In the present study, we demonstrated that a sub-MIC of AZM showed strong preventive activity against flagellar filament formation of the macrolide-resistant strain of serovar Typhimurium without suppressing flagellar biosynthesis. The findings from this study will provide a foundation for the elucidation of new actions of AZM against gram-negative intracellular pathogens.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Plasmids, strains, and growth conditions. The RNA methylase (N⁶ dimethylation of a specific adenine residue in the 23S rRNA) gene (ermBC) and the ERY esterase gene (ereB) were found in the same transferable plasmid in macrolide antibiotic-resistant Escherichia coli M26 by Nakamura et al. in Japan (38). A 1.3-kbp DNA fragment containing 738 bp of ermBC (EMBL/GenBank/DDBJ accession no. AB089505) was inserted into the HindIII-BamHI sites of pKS18 (a 400-bp EcoRI-ScaI fragment deletion derivative of pACYC184 in which the CHL resistance gene is removed) to yield plasmid pKS17. Strain MS248 (resistant to nalidixic acid and ERY) was constructed by introducing pKS17 into Salmonella enterica serovar Typhimurium SR-11 3306 (15). Strain MS266 (resistant to nalidixic acid and tetracycline) was constructed by introducing pKS18 into 3306.

Bacteria were grown on L broth or L agar (Difco Laboratories, Detroit, Mich.) supplemented with antibiotics at the following concentrations: nalidixic acid, 25 µg/ml; tetracycline, 15 µg/ml; ERY, 100 µg/ml. All of these antibiotics were purchased from Sigma-Aldrich (St. Louis, Mo.). AZM, clarithromycin (CLR), roxithromycin (RXM), rokitamycin (RKM), and CHL were obtained from Pfizer Pharmaceuticals, Inc. (New York, N.Y.), Taisho Co., Ltd. (Tokyo, Japan), Eisai Co., Ltd. (Tokyo, Japan), Asahi Kasei Co., Ltd. (Tokyo, Japan), and Sankyo Co., Ltd. (Tokyo, Japan), respectively.

MIC. A disk diffusion test was performed to determine the MICs of the antibiotics as follows. An overnight bacterial culture grown in Mueller-Hinton broth (Difco Laboratories) was diluted in the same broth, and a final inoculum with 10⁷ CFU was applied with a microplanter (Sakuma Seisakusho Ltd., Tokyo, Japan) onto Mueller-Hinton agar plates containing twofold serial dilutions of each antibiotic. The MIC was determined after an overnight culture at 37°C (19).

Cell culture model for Salmonella infection. The RAW264.7 murine macrophage-like cell line and the Henle-407 human intestinal epithelial cell line (Intestine 407, ATCC CCL 6) were maintained in Dulbecco's modified Eagle medium (Sigma-Aldrich) containing 8% heat-inactivated fetal calf serum (Gibco BRL, Grand Island, N.Y.), 50 µM ß-mercaptoethanol (Kanto Chemical Co. Inc., Tokyo, Japan), penicillin (100 U/ml; Banyu Pharmaceutical Co. Ltd., Tokyo, Japan), and streptomycin (100 µg/ml; Meiji Seika Kaisha Ltd., Tokyo, Japan). Cell cultures were seeded at a density of 1 x 10⁵ cells per well in 24-well tissue culture plates (Becton Dickinson Labware, Franklin Lakes, N.J.) with antibiotic-free culture medium 18 h prior to bacterial infection. The cell monolayers were rinsed three times with Hanks' balanced salt solution (HBSS; Sigma-Aldrich) to remove the antibiotics completely before bacterial infection (54).

For the assay of survival in macrophages, exponential-phase salmonellae were opsonized with an equal volume of mouse serum by incubation for 15 min at 37°C prior to infection of RAW264.7 cells at a multiplicity of infection (MOI) of 10 bacteria per host cell. After infection for 1 h at 37°C, infected monolayers were gently washed three times with 1 ml HBSS and incubated for 1 h at 37 °C in fresh medium containing 100 µg/ml gentamicin (Gibco BRL) to kill the extracellular bacteria. After 1-h incubation, the monolayers were washed three times with HBSS and incubated in fresh medium containing 10 µg/ml gentamicin for the remainder of the experiment. To harvest intracellular bacteria, the monolayers were washed three times with HBSS and lysed by vigorous aspiration with 1 ml phosphate-buffered saline, pH 7.4, containing 0.1% (wt/vol) sodium deoxycholate (33). The intracellular CFU of salmonellae were enumerated by diluting the lysed cells with phosphate-buffered saline containing 0.01% (wt/vol) gelatin (BSG) and plating them using triplicate sampling of each infected well.

For the assay of invasion of epithelial cells, Henle-407 cells were infected with exponential-phase salmonellae at an MOI of 100 bacteria per host cell. When necessary, a mild centrifugal force (600 x g for 10 min) was applied to the 24-well tissue culture plates at the start of the infection period. After infection for 1 h at 37°C, the infected monolayers were gently washed three times with 1 ml HBSS to remove noninvasive bacteria and incubated for 1 h in fresh medium containing gentamicin (100 µg/ml) to kill the extracellular bacteria (48, 54). After 1-h incubation, the monolayers were washed three times with HBSS, and the intracellular bacteria were harvested to enable enumeration of the bacterial CFU as described above.

Swarming and swimming motility assays. Phenotype assays for swarming and swimming were initiated by spotting 2 µl of an overnight culture at the center of brain heart infusion agar (Difco Laboratories) plates containing 0.7% agar to evaluate swarming motility and 0.3% agar to evaluate swimming motility (22, 23). The plates were analyzed after 16 h of incubation at room temperature.

Transmission electron microscopy. Salmonellae were grown in L broth supplemented with or without AZM (10 µg/ml) for more than 12 h at 37 °C without shaking. Bacterial cells were deposited on the grids, fixed with 1.0% formaldehyde for 1 h, washed with distilled water, and stained with 0.5% phosphotungstic acid. These negatively stained samples were viewed on a transmission electron microscope (JEM1010; JEOL Ltd., Tokyo, Japan) at an 80-kV accelerating voltage as described before (50).

Collection of secreted and accumulated flagellin. The cultivated salmonellae were centrifuged at 12,500 x g for 10 min at 4°C. To prepare secreted proteins, the supernatant was filtered through a Millex-GV filter (Millipore Co., Bedford, Mass.). The filtrates were mixed with prechilled trichloroacetic acid (TCA; final concentration, 10%), chilled on ice for 15 min, and centrifuged at 12,500 x g for 10 min at 4°C. The pellets were washed once with 5% TCA and twice with acetone (50).

From the pooled bacterial cells, flagellin (FliC) protein was prepared according to a laboratory manual entitled Methods in Practical Laboratory Bacteriology (7). In brief, the bacterial cell harvest was suspended in 1.5 ml of 0.15 M sodium chloride and incubated for 30 min at 60°C. After centrifugation (12,500 x g for 10 min at 4°C), approximately 300 µl of the supernatant including FliC was pooled and stored at –20 °C until required. The concentration of protein was determined by using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, Calif.) with bovine serum albumin (Sigma-Aldrich) as the standard.

Immunoblot analysis. To confirm the amount of FliC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by Western immunoblot analysis, was performed as described previously (31). The modifications made in this study were as follows. The primary and secondary antibodies used here were a monoclonal antibody to E. coli FliC (clone 15D8; IGEN International Inc., Gaithersburg, Md.) and horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (Southern Biotechnology Associates Inc., Birmingham, Ala.), used at dilutions of 1:1,000 and 1:6,000, respectively. The immunoblot detection system used was the Western Lightning Chemiluminescence Reagent Plus kit (Perkin-Elmer Inc., Wellesley, Mass.) and the VersaDoc 3000 charge-coupled device camera set (Bio-Rad Laboratories). For statistical analysis, the intensities of protein bands generated by immunostaining in Western blot assays were determined by using NIH Image version 1.6.1.

Mouse model of Salmonella infection. Seven-week-old female BALB/c mice (Charles River Japan Inc., Yokohama, Japan) were orally inoculated with 2 x 10⁸ CFU of exponential-phase salmonellae (the optical density at 600 nm reached 0.3 to 0.4) concentrated in 0.02 ml with BSG. The spleen, Peyer's patches (PP), mesenteric lymph nodes (MLN), and cecum were removed, homogenized with BSG, and plated in order to determine the number of CFU (16, 33, 34).

Statistics. Significant differences between the means plus or minus the standard deviations (SD) of different groups were examined using a two-tailed Student t test. A P value of <0.05 was regarded as statistically significant.

Top Abstract Introduction Materials and Methods Results Discussion References

 
MICs of macrolide antibiotics for serovar Typhimurium. The ermBC gene used in the present study was almost identical to the ermBC gene previously reported (3), except for a single base change, which caused replacement of the 61st amino acid, Ser (AGT [the amino acid in question is underlined]), with Asn (AAT) in the ermBC gene used in this study. Three isogenic strains, derived from serovar Typhimurium SR-11, were prepared as described in Materials and Methods: 3306 (parent strain), MS248 (ermBC introduced into 3306), and MS266 (vector introduced into 3306). The MIC of each macrolide antibiotic (AZM, CLR, ERY, RXM, or RKM) for the three strains was as follows: MS248, >100 µg/ml of every macrolide; 3306 and MS266, 100 µg/ml of every macrolide except for AZM, 6.25 µg/ml of AZM. We then analyzed AZM's efficacy in reducing the virulence function of serovar Typhimurium.

Intracellular potency of AZM against serovar Typhimurium proliferation within a mouse macrophage-like cell line. Serovar Typhimurium, described as a facultative intracellular parasite, can survive and replicate within host phagocytic cells (macrophages). The mouse macrophage-like cell line RAW264.7 can readily be infected with 3306 (47), and we determined the intracellular activity of AZM against serovar Typhimurium in this line of cells. Cells were infected with 3306, MS248, or MS266 (MOI, 10) for 1 h at 37 °C prior to exchange of the cell culture medium supplemented with AZM (10 µg/ml) or not supplemented. As shown in Fig. 1, when the cell culture medium lacked AZM, the numbers of intracellular salmonellae (CFU) among 3306, MS248, and MS266 recovered from RAW264.7 cells were constant for 24 h after infection. However, when the cell culture medium contained AZM, the bacterial numbers of these three strains recovered from RAW264.7 cells were lower at 3 h after infection than at hour 0. At hour 24 after infection, the bacterial numbers of 3306 and MS266 recovered from RAW264.7 cells had decreased dramatically from hour 0, in contrast to the constant number of MS248 recovered from RAW264.7 cells. Thus, it was concluded that AZM, at a dose of 10 µg/ml, was unable to kill MS248 at all within the macrophages.

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FIG. 1. Effect of AZM on the intracellular colonization of serovar Typhimurium within RAW264.7 cells. Monolayers (1 x 105 cells/well) were infected for 1 h with 1 x 106 CFU of opsonized 3306, MS248, or MS266. After infection, the cell culture medium was exchanged for fresh medium containing gentamicin (100 µg/ml) with or without AZM (10 µg/ml) (at hour 0). One hour later, the cell culture medium was exchanged again for fresh medium containing gentamicin (10 µg/ml) with or without AZM (10 µg/ml). The intracellular CFU of salmonellae was counted at hours 0, 3, and 24 after infection. Data represent the mean ± SD of three experiments with triplicate assays. Solid circles, 3306 without AZM; open circles, 3306 with AZM; solid squares, MS248 without AZM; open squares, MS248 with AZM; solid triangles, MS266 without AZM; open triangles, MS266 with AZM. *1, P = 0.005; *2, P = 0.001; *3, P = 0.003; *4, P = 0.2; *5, P = 0.006; *7, P = 0.0004 (compared with each strain without AZM at each point).

Extracellular potency of AZM against serovar Typhimurium invasion of an epithelial cell line. Another important virulence factor of serovar Typhimurium is its ability to enter the intestinal epithelium. Therefore, we examined the inhibitory effects of AZM on the invasion and replication of serovar Typhimurium in Henle-407 intestinal epithelial cells. The cells were infected with 3306, MS248, or MS266 (MOI, 100) for 1 h at 37 °C in cell culture medium supplemented with AZM (10 µg/ml) or not supplemented. Figure 2 shows that AZM was effective at suppressing the invasion of 3306 or MS266 into this line of cells (the invasion rate was no more than 1% of that in untreated controls). However, AZM had a small but distinct effect on the suppression of bacterial invasion in MS248 (P = 0.0002). These results led to the question of whether or not these antibiotics might be able to exert an effect on the replication of bacteria within the cells. If such an effect existed, then the data might not be solely reflective of the efficiency of bacterial invasion. Hence, we examined the ability of 3306, MS248, or MS266 to replicate within Henle-407 cells in the presence of AZM. After the cells were infected with salmonellae in the medium without AZM for 1 h, the cell culture medium was exchanged for a medium containing AZM (10 µg/ml). The numbers of 3306, MS248, and MS266 salmonellae remained constant for at least 4 h within the cells, indicating that AZM did not exert an effect on the replication rate within the cells under these experimental conditions (data not shown). Thus, it was concluded that AZM, at a concentration of 10 µg/ml, decreased the entry of MS248 into Henle-407 cells to a certain extent. As shown in Fig. 2, when salmonellae invaded Henle-407 cells in AZM-containing culture medium, centrifugation was required for sufficient numbers of bacteria to enter the cells. Consequently, we concluded that AZM at a sub-MIC might have inhibited the motility of bacteria.

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FIG. 2. Effect of AZM on the invasion of Henle-407 cells by serovar Typhimurium. Monolayers (1 x 105 cells/well) were infected for 1 h with 1 x 107 CFU of 3306, MS248, or MS266 in the presence or absence of AZM (10 µg/ml) with or without a centrifugal force at the start of the infection period. After 1 h infection, the cell culture medium was exchanged for fresh medium containing gentamicin (100 µg/ml). One hour later, intracellular CFU of salmonellae were counted. Data represent the mean ± SD of three experiments with triplicate assays. Open columns, 3306; filled columns, MS248; dotted columns, MS266.

Effects of macrolides on the swarming or swimming motility of MS248. We examined the effects of AZM and other macrolide antibiotics on the motility of salmonellae. As shown in Fig. 3, MS248 hardly exhibited swarming (Fig. 3A, no. 2) or swimming (Fig. 3B, no. 2) motility in the presence of AZM (10 µg/ml). In contrast, MS248 exhibited swarming motility in the presence of ERY, CLR, RXM, or RKM (10 µg/ml), as shown in Fig. 3A, no. 3, no. 4, no. 5, or no. 6, respectively. However, ERY and CLY reduced the swimming motility of MS248 in part, as shown in Fig. 3B, no. 3 and no. 4, respectively. Therefore, among these macrolide antibiotics, only AZM (10 µg/ml) was able to limit both the swarming and swimming motilities of MS248.

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FIG. 3. Swarming and swimming motilities in MS248. Swarming and swimming plates were made of brain heart infusion with 0.7% (A) and 0.3% (B) agar, respectively, supplemented with AZM (no. 2), ERY (no. 3), CLR (no. 4), RXM (no. 5), or RKM (no. 6) at a concentration of 10 µg/ml or without antibiotic (no. 1). After inoculation with MS248, plates were incubated for 16 h at room temperature.

Effects of AZM on the morphological changes of MS248. To determine how AZM limits the motilities of MS248, we examined the morphological changes of bacterial cells with AZM by electron microscopic analysis. MS248 was grown overnight at 37 °C as static cultures in L broth supplemented with AZM (10 µg/ml) or not supplemented. Electron micrographs showed no flagellar filaments on the bacterial surface at all when the L broth contained AZM (Fig. 4B), whereas large numbers of flagellar filaments were seen when no AZM was added to the culture medium (Fig. 4A). In contrast, fimbrial structures were observed on the surface of salmonellae in either the presence or the absence of AZM. These results indicated that AZM, even at a sub-MIC, inhibited the flagellar filament formation of MS248.

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FIG. 4. Transmission electron microscopy of MS248. Salmonellae were grown in L broth supplemented with AZM (10 µg/ml) (B) or without antibiotic supplementation (A) for more than 12 h without shaking at 37°C. Bars, 2 µm.

Effects of AZM on the flagellar formation of MS248. We examined the effects of AZM at different concentrations (0, 5, and 10 µg/ml) on the growth rate and protein synthesis of MS248. The rate of protein synthesis was measured by the method of pulse-labeling with [³⁵S]methionine-cysteine as previously described by Yamanaka et al. (56). In brief, aliquot samples (400 µl) from cultures were removed, added to tubes containing [³⁵S]methionine-cysteine (3.7 x 10⁶ Bq), and incubated for 3 min at 37°C. Fifty microliters of each sample was spotted onto 3MM filter paper. The filters were soaked in ice-cold 5% TCA and rinsed with ice-cold ethanol. Dried filters were counted in a liquid scintillation counter. The growth curve patterns of MS248 in L broth containing different concentrations of AZM were much the same (data not shown). In addition, at each time point (2, 4, 6, and 8 h), the rate of incorporation of [³⁵S]methionine-cysteine into bacterial cells of MS248 was also the same in medium with AZM (5 or 10 µg/ml) and medium without it (data not shown). These findings demonstrated that AZM at a concentration of 10 µg/ml had no inhibitory effect on the replication rate or total protein synthesis of MS248.

Since a flagellar filament consists of an assembly of about 20,000 subunits of a single protein, flagellin (FliC) (30), we examined the effects of AZM on the specific synthesis of FliC protein and/or on the export of FliC from bacterial cells. At the 8-h time point of the MS248 cultures mentioned above, FliC and total secreted proteins were extracted from whole bacterial cells and culture medium, respectively. First, the total proteins secreted by MS248 into L broth were collected by TCA, separated by SDS-PAGE, stained with Coomassie brilliant blue (CBB), and identified according to the description of Komoriya et al. (24). As shown in Fig. 5A, the major proteins secreted by MS248 were identified as either flagellar components, including FlgK (58 kDa), FliC (52 kDa), FliD (50 kDa), FlgE (42 kDa), FlgL (35 kDa), and FlgD (28 kDa), or virulence factors, including SipA (89 kDa), SipB (67 kDa), and SipC (42 kDa). Neither ERY, nor CLR, nor RXM (10 µg/ml) reduced the intensities of these protein bands at all, as shown in lanes 2, 3, and 4 (Fig. 5A). By comparison, AZM (10 µg/ml) greatly reduced the intensity of the FliC protein band (FliC is the most abundant of the exported proteins), as shown in lane 5 (Fig. 5A). We therefore examined the changes in the amounts of FliC inside and outside bacterial cells of MS248 by Western blotting with immunostaining. As shown in Fig. 5B, no differences were found in the amounts of FliC extracted from whole bacterial cells of MS248 between the medium supplemented with AZM (5 or 10 µg/ml; lane 2 or 3, respectively) and not supplemented (lane 1) in the immunostained protein bands. In contrast, Fig. 5C shows a 70% decrease in the amount of FliC in the medium supplemented with AZM (5 or 10 µg/ml; lane 2 or 3, respectively) compared with the AZM-free medium (lane 1). These findings indicate that AZM (5 or 10 µg/ml) did not affect the synthesis of flagellin within the bacterial cells but it mainly decreased flagellar export from MS248 bacterial cells.

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FIG. 5. SDS-PAGE and immunoblot analyses of secreted proteins from MS248. (A) After 8 h of standing cultivation, the proteins secreted by MS248 bacterial cells were separated by 10% SDS-PAGE and stained with CBB. Ten micrograms of protein was applied to each lane. Lanes: 1, without antibiotic in the medium; 2, with ERY (10 µg/ml) in the medium; 3, with CLR (10 µg/ml) in the medium; 4, with RXM (10 µg/ml) in the medium; 5, with AZM (10 µg/ml) in the medium; M, molecular mass markers. (B, C) The FliC protein bands from whole-cell lysates (B) or from supernatants (C) were detected by Western blotting with immunostaining as described in Materials and Methods. One microgram of protein was applied to each lane of a 12% SDS-PAGE analysis. Lanes: 1, without antibiotic in the medium; 2, with AZM (5 µg/ml) in the medium; 3, with AZM (10 µg/ml) in the medium. Molecular masses are shown. Arrows indicate FliC protein bands.

Clearance of serovar Typhimurium from tissues of AZM-treated mice. We examined AZM's efficacy at curing murine salmonellosis. We repeated the intraperitoneal administration of AZM (20 mg/kg body weight) for 3 days (once a day) from day 3 to day 5 after oral inoculation with 2 x 10⁸ CFU of 3306 or MS248. At day 6 after infection, the number of bacterial CFU in each tissue was measured by plating. As shown in Fig. 6, AZM was clearly effective in the treatment of murine salmonellosis caused by 3306, since there were statistically significant differences in the rates of bacterial recovery from every tissue except for PP (P > 0.1) between control and AZM-treated mice (Fig. 6A). By comparison, there were no statistically significant differences between control and AZM-treated mice in the recovery rates of MS248 bacteria from the spleen and PP (Fig. 6B). In the MLN, the difference between control and AZM-treated mice was marginal (P < 0.05) (Fig. 6B). In the cecum, the recovery rate of MS248 from AZM-treated mice was significantly greater than that from control mice (Fig. 6B). When mice were not administered AZM, there were no differences between 3306 and MS248 in the bacterial recovery rates from any of the tissue samples. These findings indicate that, as a treatment against murine salmonellosis caused by MS248 as opposed to that caused by 3306, AZM was ineffective under these experimental conditions.

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FIG. 6. Efficacy of AZM against the colonization of mice by serovar Typhimurium. Mice were orally inoculated with 2 x 108 CFU of 3306 or MS248. From day 3 to day 5 postinoculation, mice received daily intraperitoneal administration of AZM (20 mg/kg body weight) at the same time each day. On day 6, the cecum, PP, MLN, and spleen were examined for CFU of 3306 (A) or MS248 (B). (A) Open columns, 3306 infected without AZM treatment; filled columns, 3306 infected and AZM received. (B) Dotted columns, MS248 infected without AZM treatment; filled columns, MS248 infected and AZM received. Data represent the mean ± SD of two experiments. Each group had six mice.

Top Abstract Introduction Materials and Methods Results Discussion References

 
It has been generally recognized that the use of certain concentrations of macrolide antibiotics, including AZM, ERY, and CLR, on a long-term treatment basis is effective against chronic lung disease in cystic fibrosis, where Pseudomonas aeruginosa is the main pathogen (45). As a matter of fact, it was previously shown that some macrolide antibiotics containing AZM at sub-MICs were able to inhibit the motility of Proteus mirabilis and P. aeruginosa, owing to the decline of flagellar expression (20, 21, 37). Similarly, Horii et al. reported that sub-MICs of mupirocin reduced the expression of flagellin in P. mirabilis and P. aeruginosa (17). In Salmonella, by comparison, we demonstrated that although the macrolide antibiotics at sub-MICs could not suppress flagellar expression, a sub-MIC of AZM nevertheless reduced flagellar export (Fig. 5). Presumably, then, a sub-MIC of AZM might affect the export apparatus involved in flagellar assembly.

It is well known that the flagellar operons are divided into three classes with respect to their transcription hierarchy (26). Class 3 contains five operons, including a filament formation operon. In addition, most Salmonella serovars have two genes for a major component protein of the filament at different locations on the chromosome that code for the antigenically distinct flagellar types, H1 (phase 1 [FliC]) and H2 (phase 2 [FljB]) (26, 30). The expression of the class 3 operon requires FliA (class 3 operon-specific sigma factor ²⁸). The fliA gene is included in class 2, and it has been found to positively regulate expression by activator proteins FlhD and FlhC, which are encoded by the flhD class 1 operon lying at the top of the transcription hierarchy (27, 29). Previous studies indicated the importance of flagella for Salmonella invasion of cultured cells. Strains deficient in the flagellar transcriptional regulators flhDC and fliA were unable to fully invade Henle-407 cells even after the application of mild centrifugal force to the tissue culture plates (11). Since this result is consistent with our findings (Fig. 2), we presume that the loss of flagellar filaments impeded the invasion of MS248 in the presence of AZM (10 µg/ml).

Komoriya et al. precisely analyzed all of the major proteins secreted into culture medium by serovar Typhimurium. They detected protein bands of 89, 67, 58, 52, 50, 42, 40, 35, and 28 kDa, corresponding to SipA, SipB, FlgK, FliC, FliD, a mixture of SipC and FlgE, InvJ, FlgL, and FlgD, respectively, through SDS-PAGE with CBB staining (24). SipA, SipB, SipC, and InvJ were specific for virulence factors as described below, and the other six proteins were specific for flagellar components (2, 24). In the present study, SDS-PAGE analysis identified four protein bands, FlgE (hook), FlgK (hook-filament junction at hook), FlgL (hook-filament junction at filaments), and FliD (filament cap), of secreted flagellar components in culture medium supplemented with AZM (Fig. 5A). There are two types of export specificity, the rod-hook type and the filament type (2). Since AZM (10 µg/ml) greatly reduced the export of FliC only from MS248 bacterial cells (Fig. 5B), we assume that AZM (10 µg/ml) affected the export of the filament type but not of the rod-hook type during the cultivation of MS248. Flagellar filaments are reconstituted by polymerization with exported flagellin monomers at the tips of normal hooks on the bacterial cells (2, 24). In the presence of AZM (10 µg/ml), no flagellar filaments were observed on the surface of bacterial cells in MS248 (Fig. 4B) despite the fact that about 30% of flagellin proteins still existed in the culture medium (Fig. 5C). This indicates that a sub-MIC of AZM might suppress the polymerization of flagellin monomers, as well as flagellar export.

Another important component of the virulence mechanisms of gram-negative pathogens is the direct translocation of bacterial effector proteins into the cytoplasm of eukaryotic host cells by specialized secretion systems, termed type III secretion systems (TTSSs). TTSSs are composed of more than 20 highly conserved proteins encoded on plasmids and large chromosomal pathogenicity islands (18, 40, 44). Salmonella pathogenicity island 1 (SPI1) and SPI2 are located at 63 and 30 centisomes, respectively, on the chromosome. The TTSSs encoded on SPI1 are required for bacterium-mediated invasion of host cells (9, 13), and the TTSSs encoded on SPI2 are required for bacterial replication within host macrophages (8, 39). Both SPI1 and SPI2 have two separate TTSS structural components, which secrete proteins from the bacterium and occasionally into host cells. With the exception of the flagellar system, the needle complex of TTSSs looks strikingly similar to the flagellar hook-basal body complex protein (25). In fact, at least seven components of the flagellar hook-basal body complex protein are homologous to a subset of components that make up the TTSSs (28). Therefore, it is thought possible that AZM might be involved in a specific inhibitory effect on TTSSs. Figure 5A shows a slight reduction of SipA secretion by a sub-MIC of AZM. However, it shows no reduction of SipB or SipC secretion by AZM treatment. Other secreted proteins involved in the SPI-1 TTSS could not be analyzed by this SDS-PAGE method. Based on these findings, we suppose that a sub-MIC of AZM did not affect the SPI-1 TTSS in MS248.

A recent report indicated that a sub-MIC (2 µg/ml) of AZM interfered with the synthesis of autoinducers (AIs) such as 3-oxo-C₁₂-homoserine lactone and N-butyryl-L-homoserine lactone in a quorum-sensing system, leading to a reduction in virulence factors in P. aeruginosa (49). Further investigation indicated that AZM repressed the expression of the AI synthase genes lasI and rhlI by an unknown mechanism (49). In contrast, serovar Typhimurium and serovar Typhi produce and detect the second AI (AI-2). AI-2 is produced by a remarkably wide variety of gram-negative and gram-positive bacteria, and its production requires a protein called LuxS in every case. In addition, AI-2 (furanones) is a universal signal that functions in interspecies cell-to-cell communication (1, 36, 55). It remains unknown whether or not a sub-MIC of AZM interferes with the synthesis of AI-2 in MS248. It will be very important to obtain this knowledge.

What is most important to consider is that AZM's efficacy in interfering with the formation of flagellar filaments does not directly lead to bactericidal potency against MS248 in BALB/c mice (Fig. 6). We previously reported that in an oral inoculation trial, a large quantity of salmonellae (more than 1 x 10³ CFU) already resided inside the macrophages in the spleen at day 3 after infection (32). Supposedly, flagella were unnecessary for salmonellae to proliferate within the macrophages at this point. Actually, a sub-MIC of AZM was unable to lead to bactericidal efficacy against MS248 within RAW264.7 cells (Fig. 1). This result supports our speculation. In the present study, AZM was administered from day 3 to day 5. But by then it may have been too late for AZM to work against murine salmonellosis caused by MS248. There remains a possibility that a sub-MIC of AZM diminishes the adhesion and invasion steps of MS248 in the small intestines of mice. Still, it is an important challenge to reveal whether or not AZM treatment is effective against murine salmonellosis caused by MS248.

 
We are grateful to Kazuhiro Tateda and Hajime Hashimoto (Toho University) for critical reading of the manuscript and for helpful suggestions.

This work was supported in part by a Grant-in-Aid for Scientific Research C (15590398) to H.M. and in part by a grant from the 21st Century COE Program to S.O. from the Ministry of Education, Culture, Sports, Science, and Technology of the Japanese Government. This work was also supported in part by a subsidy for research into Salmonella pathogenesis to H.M. from Pfizer Pharmaceuticals, Inc.

 
* Corresponding author. Mailing address: Laboratory of Immunoregulation, Department of Infection Control and Immunology, Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan. Phone and fax: 81-3-5791-6267. E-mail: hmatsui{at}lisci.kitasato-u.ac.jp.

Present address: Department of Microbiology, School of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama 350-0295, Japan.

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Ahmer, B. M. 2004. Cell-to-cell signaling in Escherichia coli and Salmonella enterica. Mol. Microbiol. 52:933-945.[CrossRef][Medline]
2
2. Berg, H. C. 2003. The rotary motor of bacterial flagella. Annu. Rev. Biochem. 72:19-54.[CrossRef][Medline]
3
3. Brisson-Noel, A., M. Arthur, and P. Courvalin. 1988. Evidence for natural gene transfer from gram-positive cocci to Escherichia coli. J. Bacteriol. 170:1739-1745.[Abstract/Free Full Text]
4
4. Brumell, J. H., and B. B. Finlay. 1999. Bacterial adherence, colonization, and invasion of mucosal surfaces, p. 3-17. In K. A. Brogden, J. A. Roth, T. B. Stanton, C. A. Bolin, F. C. Minion, and M. J. Wannemuehler (ed.), Virulence mechanisms of bacterial pathogenesis. American Society for Microbiology, Washington, D.C.
5
5. Butler, T. 2001. Effect of increased inoculum of Salmonella typhi on MIC of azithromycin and resultant growth characteristics. J. Antimicrob. Chemother. 48:903-906.[Abstract/Free Full Text]
6
6. Butler, T. 2002. Reduction of MIC of azithromycin for Salmonella typhi and Staphylococcus aureus in broth exposed to previous growth of Salmonella typhi. J. Antimicrob. Chemother. 50:143-145.[Free Full Text]
7
7. Chart, H. 1994. Bacterial fractionation and membrane protein characterization, p. 1-10. In H. Chart (ed.), Methods in practical laboratory bacteriology. CRC Press, Inc., Boca Raton, Fla.
8
8. Cirillo, D. M., R. H. Valdivia, D. M. Monack, and S. Falkow. 1998. Macrophage-dependent induction of the Salmonella pathogenicity island 2 type III secretion system and its role in intracellular survival. Mol. Microbiol. 30:175-188.[CrossRef][Medline]
9
9. Darwin, K. H., and V. L. Miller. 1999. Molecular basis of the interaction of Salmonella with the intestinal mucosa. Clin. Microbiol. Rev. 12:405-428.[Abstract/Free Full Text]
10
10. Duran, J. M., and G. W. Amsden. 2000. Azithromycin: indications for the future? Expert Opin. Pharmacother. 1:489-505.[CrossRef][Medline]
11
11. Eichelberg, K., and J. E. Galan. 2000. The flagellar sigma factor FliA (²⁸) regulates the expression of Salmonella genes associated with the centisome 63 type III secretion system. Infect. Immun. 68:2735-2743.[Abstract/Free Full Text]
12
12. Frenck, R. W., Jr., I. Nakhla, Y. Sultan, S. B. Bassily, Y. F. Girgis, J. David, T. C. Butler, N. I. Girgis, and M. Morsy. 2000. Azithromycin versus ceftriaxone for the treatment of uncomplicated typhoid fever in children. Clin. Infect. Dis. 31:1134-1138.[CrossRef][Medline]
13
13. Galan, J. E., and D. Zhou. 2000. Striking a balance: modulation of the actin cytoskeleton by Salmonella. Proc. Natl. Acad. Sci. USA 97:8754-8761.[Abstract/Free Full Text]
14
14. Gulig, P. A. 1996. Pathogenesis of systemic diseases, p. 2774-2787. In F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed. American Society for Microbiology, Washington, D.C.
15
15. Gulig, P. A., and R. Curtiss III. 1987. Plasmid-associated virulence of Salmonella typhimurium. Infect. Immun. 55:2891-2901.[Abstract/Free Full Text]
16
16. Gulig, P. A., and T. J. Doyle. 1993. The Salmonella typhimurium virulence plasmid increases the growth rate of salmonellae in mice. Infect. Immun. 61:504-511.[Abstract/Free Full Text]
17
17. Horii, T., M. Morita, H. Muramatsu, Y. Muranaka, T. Kanno, and M. Maekawa. 2003. Effects of mupirocin at subinhibitory concentrations on flagella formation in Pseudomonas aeruginosa and Proteus mirabilis. J. Antimicrob. Chemother. 51:1175-1179.[Abstract/Free Full Text]
18
18. Hueck, C. J. 1998. Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol. Mol. Biol. Rev. 62:379-433.[Abstract/Free Full Text]
19
19. Japan Society of Chemotherapy. 1981. Method of MIC determination. Chemotherapy 29:76-79.
20
20. Kawamura-Sato, K., Y. Iinuma, T. Hasegawa, T. Horii, T. Yamashino, and M. Ohta. 2000. Effect of subinhibitory concentrations of macrolides on expression of flagellin in Pseudomonas aeruginosa and Proteus mirabilis. Antimicrob. Agents Chemother. 44:2869-2872.[Abstract/Free Full Text]
21
21. Kawamura-Sato, K., Y. Iinuma, T. Hasegawa, T. Yamashino, and M. Ohta. 2001. Postantibiotic suppression effect of macrolides on the expression of flagellin in Pseudomonas aeruginosa and Proteus mirabilis. J. Infect. Chemother. 7:51-54.[CrossRef][Medline]
22
22. Kim, W., T. Killam, V. Sood, and M. G. Surette. 2003. Swarm-cell differentiation in Salmonella enterica serovar Typhimurium results in elevated resistance to multiple antibiotics. J. Bacteriol. 185:3111-3117.[Abstract/Free Full Text]
23
23. Kim, W., and M. G. Surette. 2003. Swarming populations of Salmonella represent a unique physiological state coupled to multiple mechanisms of antibiotic resistance. Biol. Proc. Online 5:189-196.
24
24. Komoriya, K., N. Shibano, T. Higano, N. Azuma, S. Yamaguchi., and S. I. Aizawa. 1999. Flagellar proteins and type III-exported virulence factors are the predominant proteins secreted into the culture media of Salmonella typhimurium. Mol. Microbiol. 34:767-779.[CrossRef][Medline]
25
25. Kubori, T., Y. Matsushima, D. Nakamura, J. Uralil, M. Lara-Tejero, A. Sukhan, J. E. Galan, and S. I. Aizawa. 1998. Supramolecular structure of the Salmonella typhimurium type III protein secretion system. Science 280:602-605.[Abstract/Free Full Text]
26
26. Kutsukake, K., Y. Ohya, and T. Iino. 1990. Transcriptional analysis of the flagellar regulon of Salmonella typhimurium. J. Bacteriol. 172:741-747.[Abstract/Free Full Text]
27
27. Kutsukake, K., Y. Ohya, S. Yamaguchi, and T. Iino. 1988. Operon structure of flagellar genes in Salmonella typhimurium. Mol. Gen. Genet. 214:11-15.[CrossRef][Medline]
28
28. Lee, A. C. 2003. Type III secretion systems of enteric bacterial pathogens, p. 403-422. In G. A. Hecht (ed.), Microbial pathogenesis and the intestinal epithelial cell. American Society for Microbiology, Washington, D.C.
29
29. Liu, X., and P. Matsumura. 1994. The FlhD/FlhC complex, a transcriptional activator of the Escherichia coli flagellar class II operons. J. Bacteriol. 176:7345-7351.[Abstract/Free Full Text]
30
30. Macnab, R. M. 1996. Flagella and motility, p. 123-145. In F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed. American Society for Microbiology, Washington, D.C.
31
31. Matsui, H., A. Abe, S. Suzuki, M. Kijima, Y. Tamura, M. Nakamura, K. Kawahara, and H. Danbara. 1993. Molecular mechanism of the regulation of expression of plasmid-encoded mouse bacteremia (mba) genes in Salmonella serovar Choleraesuis. Mol. Gen. Genet. 236:219-226.[CrossRef][Medline]
32
32. Matsui, H., M. Eguchi, and Y. Kikuchi. 2000. Use of confocal microscopy to detect Salmonella typhimurium within host cells associated with Spv-mediated intracellular proliferation. Microb. Pathog. 29:53-59.[CrossRef][Medline]
33
33. Matsui, H., T. Kawakami, S. Ishikawa, H. Danbara, and P. A. Gulig. 2000. Constitutively expressed phoP inhibits mouse-virulence of Salmonella typhimurium in an Spv-dependent manner. Microbiol. Immunol. 44:447-454.[Medline]
34
34. Matsui, H., M. Suzuki, Y. Isshiki, C. Kodama, M. Eguchi, Y. Kikuchi, K. Motokawa, A. Takaya, T. Tomoyasu, and T. Yamamoto. 2003. Oral immunization with ATP-dependent protease-deficient mutants protects mice against subsequent oral challenge with virulent Salmonella enterica serovar Typhimurium. Infect. Immun. 71:30-39.[Abstract/Free Full Text]
35
35. Maurin, M., and D. Raoult. 1997. Intracellular organisms. Int. J. Antimicrob. Agents 9:61-70.[CrossRef][Medline]
36
36. Miller, M. B., and B. L. Bassler. 2001. Quorum sensing in bacteria. Annu. Rev. Microbiol. 55:165-199.[CrossRef][Medline]
37
37. Molinari, G., P. Paglia, and G. C. Schito. 1992. Inhibition of motility of Pseudomonas aeruginosa and Proteus mirabilis by subinhibitory concentrations of azithromycin. Eur. J. Clin. Microbiol. Infect. Dis. 11:469-471.[CrossRef][Medline]
38
38. Nakamura, A., K. Nakazawa, I. Miyakozawa, S. Mizukoshi, K. Tsurubuchi, M. Nakagawa, K. O'Hara, and T. Sawai. 2000. Macrolide esterase-producing Escherichia coli clinically isolated in Japan. J. Antibiot. (Tokyo) 53:516-524.[Medline]
39
39. Ochman, H., F. C. Soncini, F. Solomon, and E. A. Groisman. 1996. Identification of a pathogenicity island required for Salmonella survival in host cells. Proc. Natl. Acad. Sci. USA 93:7800-7804.[Abstract/Free Full Text]
40
40. Ohl, M. E., and S. I. Miller. 2001. Salmonella: a model for bacterial pathogenesis. Annu. Rev. Med. 52:259-274.[CrossRef][Medline]
41
41. Posho, L., L. Delbos-Bocage, D. Gueylard, R. Farinotti, and C. Carbon. 1998. Effects of Salmonella typhimurium infection and ofloxacin treatment on glucose and glutamine metabolism in Caco-2/TC-7 cells. Antimicrob. Agents Chemother. 42:2950-2955.[Abstract/Free Full Text]
42
42. Rakita, R. M., K. Jacques-Palaz, and B. E. Murray. 1994. Intracellular activity of azithromycin against bacterial enteric pathogens. Antimicrob. Agents Chemother. 38:1915-1921.[Abstract/Free Full Text]
43
43. Scherer, C. A., and S. I. Miller. 2001. Molecular pathogenesis of Salmonella, p. 265-333. In E. A. Groisman (ed.), Principles of bacterial pathogenesis. Academic Press, San Diego, Calif.
44
44. Schmidt, H., and M. Hensel. 2004. Pathogenicity islands in bacterial pathogenesis. Clin. Microbiol. Rev. 17:14-56.[Abstract/Free Full Text]
45
45. Schoni, M. H. 2003. Macrolide antibiotic therapy in patients with cystic fibrosis. Swiss Med. Wkly. 133:297-301.[Medline]
46
46. Stock, I., and B. Wiedemann. 2000. Natural antibiotic susceptibility of Salmonella enterica strains. Int. J. Antimicrob. Agents 16:211-217.[CrossRef][Medline]
47
47. Takaya, A., M. Suzuki, H. Matsui, T. Tomoyasu, H. Sashinami, A. Nakane, and T. Yamamoto. 2003. Lon, a stress-induced ATP-dependent protease, is critically important for systemic Salmonella enterica serovar Typhimurium infection of mice. Infect. Immun. 71:690-696.[Abstract/Free Full Text]
48
48. Takaya, A., T. Tomoyasu, A. Tokumitsu, M. Morioka, and T. Yamamoto. 2002. The ATP-dependent Lon protease of Salmonella enterica serovar Typhimurium regulates invasion and expression of genes carried on Salmonella pathogenicity island 1. J. Bacteriol. 184:224-232.[Abstract/Free Full Text]
49
49. Tateda, K., R. Comte, J. C. Pechere, T. Kohler, K. Yamaguchi, and C. Van Delden. 2001. Azithromycin inhibits quorum sensing in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 45:1930-1933.[Abstract/Free Full Text]
50
50. Tomoyasu, T., T. Ohkishi, Y. Ukyo, A. Tokumitsu, A. Takaya, M. Suzuki, K. Sekiya, H. Matsui, K. Kutsukake, and T. Yamamoto. 2002. The ClpXP ATP-dependent protease regulates flagellum synthesis in Salmonella enterica serovar Typhimurium. J. Bacteriol. 184:645-653.[Abstract/Free Full Text]
51
51. Treadway, G. 2001. Azithromycin: a new 15-membered macrolide. Jpn. J. Antibiot. 54:70-76.
52
52. Walsh, C. 2003. Antibiotics that block bacterial protein synthesis, p. 51-70. In C. Walsh (ed.), Antibiotics actions, origins, resistance. American Society for Microbiology, Washington D.C.
53
53. Walsh, C. 2003. Antibiotics resistance by replacement or modification of the antibiotic target, p. 143-155. In C. Walsh (ed.), Antibiotics actions, origins, resistance. American Society for Microbiology, Washington, D.C.
54
54. Wilson, J. A., T. J. Doyle, and P. A. Gulig. 1997. Exponential-phase expression of spvA of the Salmonella typhimurium virulence plasmid: induction in intracellular salts medium and intracellularly in mice and cultured mammalian cells. Microbiology 143:3827-3839.[Abstract/Free Full Text]
55
55. Xavier, K. B., and B. L. Bassler. 2003. LuxS quorum sensing: more than just a numbers game. Curr. Opin. Microbiol. 6:191-197.[CrossRef][Medline]
56
56. Yamanaka, K., T. Ogura, H. Niki, and S. Hiraga. 1992. Identification and characterization of the smbA gene, a suppressor of the mukB null mutant of Escherichia coli. J. Bacteriol. 174:7517-7526.[Abstract/Free Full Text]

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