Effects of mupirocin at subinhibitory concentrations on flagella formation in Pseudomonas aeruginosa and Proteus mirabilis

Toshinobu Horii1,2,*, Motoki Morita1,3, Hideaki Muramatsu2,4, Yoshinori Muranaka5, Takashi Kanno1 and Masato Maekawa1

1 Department of Laboratory Medicine, 2 Group of Infection Control Research, 4 Division of Pharmacy, 5 Laboratory for Ultrastructure Research, Hamamatsu University School of Medicine, 1-20-1 Handa-yama, Hamamatsu 431-3192; 3 Daiichi Pure Chemicals, 3-3-1 Koyodai, Ryugasaki 301-0852, Japan

Received 18 April 2002; returned 27 August 2002; revised 25 October 2002; accepted 21 February 2003


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Colonization of Pseudomonas aeruginosa at trachea, nares and oropharynx can cause ventilator-acquired pneumonia. To identify beneficial effects of antibiotics on expression of virulence factors related to colonization by such pathogens, we evaluated the effect of mupirocin on flagella formation in P. aeruginosa and on motility and flagella formation in Proteus mirabilis. In P. aeruginosa, subinhibitory concentrations of mupirocin inhibited flagella formation, which was associated with reduced flagellin expression. In P. mirabilis, subinhibitory concentrations of mupirocin dose-dependently suppressed bacterial motility and flagella formation, again with reduced flagellin expression. Our results indicate that subinhibitory concentrations of mupirocin can suppress expression of virulence factors in P. aeruginosa and P. mirabilis.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Previous epidemiological analyses have shown that risk factors for ventilator-acquired pneumonia (VAP) include duration of mechanical ventilation, colonization of the upper respiratory tract and absence of antibiotic therapy.1 Before the diagnosis of VAP, pathogens that have colonized trachea, nares and oropharynx are recovered from cultures of tracheal secretions.1 The most common pathogens are Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae and Haemophilus species.1

Treatment with subinhibitory concentrations of some antibiotics suppresses expression of virulence factors in various Gram-negative bacteria.27 Macrolides and clindamycin inhibit biofilm formation in P. aeruginosa,3,5,7 and macrolides suppress flagellin expression in both P. aeruginosa and Proteus mirabilis.5 Recently, subinhibitory concentrations of azithromycin have been shown to interfere with synthesis of autoinducers such as 3-oxo-C12-homoserine lactone (HSL) and C4-HSL in the ‘quorum-sensing’ cell-to-cell signalling system, leading to reduction in virulence factors.6 Some reports have suggested that treatment with subinhibitory concentrations of antibiotics such as macrolides may benefit patients with P. aeruginosa infections through inhibition of biofilm formation.5,6 To date, a limited number of antibiotics are known to have beneficial effects on expression of virulence factors at subinhibitory concentrations.

To identify beneficial effects of antibiotics on expression of virulence factors related to colonization by pathogens such as P. aeruginosa, we assessed the effect of mupirocin on flagella formation in P. aeruginosa. Additionally, we evaluated the effect of mupirocin on flagella formation and motility in P. mirabilis.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains, media and culture conditions

P. aeruginosa PAO-1 and a clinical isolate of P. mirabilis HU2001-309 were used. Bacteria were stored at –70°C in heart infusion broth (Nissui Pharmaceutical, Tokyo, Japan) containing 20% glycerol. Subsequently, bacteria were inoculated on heart infusion agar plates (Nissui Pharmaceutical) and incubated at 37°C overnight.

Susceptibility testing

The antibiotic used was mupirocin (GlaxoSmithKline, Tokyo, Japan), following an incidental observation of inhibition of swarming of P. mirabilis by this agent. MICs were determined by an agar dilution method as described by the National Committee for Clinical Laboratory Standards.8 Susceptibility testing was carried out on Mueller–Hinton agar (Nippon Becton Dickinson, Tokyo, Japan) in accordance with the manufacturer’s instructions.

Flagella preparation

Bacteria were cultured on heart infusion agar with either mupirocin or no antibiotic at 37°C for 12, 16 and 20 h. Bacteria were then gently scraped from the agar surface and suspended in PBS (pH 7.4). These samples were used for preparation of flagellin protein and for morphological observation by scanning electron microscopy (SEM). Flagellin protein was prepared by the method of Kawamura-Sato et al.5,9 The bacterial suspension was centrifuged (5000g) for 15 min at 4°C. The resulting pellet was resuspended in PBS and agitated with a tube mixer for 3 min to shear off the flagella. The suspension was centrifuged (16 000g) for 15 min at 4°C, and the resulting supernatant was centrifuged (40 000g) again for 3 h at 4°C. The supernatant was carefully removed and the pellet was suspended in PBS. The sample was analysed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The total protein concentrations in these samples were determined using a Micro BCA protein assay reagent kit (Pierce, Rockford, IL, USA). Concentrations of the flagellin protein were measured by the National Institutes of Health Image program, version 1.62, which is a public domain image of processing and analysis program for the Macintosh.10 These experiments were carried out twice.

The criteria for quantifying flagella formation were defined as follows: high formation where the number and length were similar to formation with no antibiotic, moderate formation where the number was reduced but the length was preserved compared with formation with no antibiotic, and low formation where a small number of short flagella remained.

Scanning electron microscopy

Samples were fixed with Karnovsky’s fixative (2% paraformaldehyde and 2.5% glutaraldehyde in PBS, pH 7.4), stained with 1% osmium tetroxide and 1% tannic acid and then dehydrated for 10 min in each of several increasing concentrations of ethanol (50, 70, 80, 90, 95 and 100% v/v), followed by two 10 min immersions in 100% 2-methyl-2-propanol at 45°C. Samples were freeze-dried (JFD-300; Jeol, Akishima, Japan) and osmium-coated with Plasma Multi Coater PMC-5000 (Meiwa Shoji, Osaka, Japan) for 16 s. Specimens were then examined with an S-800 electron microscope (Hitachi, Tokyo, Japan) at an accelerating voltage of 10 kV.11

Motility assay

Motility was assessed as described by Kawamura-Sato et al.5 Bacteria grown in heart infusion broth at 37°C to an optical density of 0.1 at 660 nm were inoculated on to 0.3% heart infusion agar plates containing no antibiotic and 0.125, 0.25 and 0.5 x MIC of mupirocin for 18 h at 30°C. Diameters of growth zones were measured on the plates containing mupirocin or no antibiotic and were expressed as the mean ± S.D. for three experiments.

Statistical analysis

Statistical analysis was carried out using Microsoft Excel 97 Windows Edition. Results are expressed as mean ± S.D.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antibiotic susceptibility

The MIC of mupirocin was >8192 mg/L for P. aeruginosa PAO-1 and 128 mg/L for P. mirabilis HU2001-309.

Effect of mupirocin on flagella formation in P. aeruginosa

The effect of mupirocin at 64, 256 and 1024 mg/L on P. aeruginosa flagella formation was examined by SEM (Figure 1c–f). After 12 h of exposure to mupirocin, flagella formation was moderate at 1024 mg/L mupirocin, but flagella formation after exposure to both 64 and 256 mg/L mupirocin was high (data not shown). After 16 h of exposure, flagella formation was low at 1024 mg/L, moderate at 256 mg/L and high at 64 mg/L (Figure 1c–f). To examine whether the reduced flagella formation was associated with loss of flagellin expression, the flagellin fraction of P. aeruginosa was prepared. Decrease in intensity of a single major band of the flagellin protein separated on an SDS–PAGE gel was related to suppression of flagella formation according to SEM (Figure 1a). The quantitative estimates of the flagellin protein concentrations by the National Institutes of Health Image program were 108.86, 76.52, 60.98 and 40.96 for P. aeruginosa grown for 16 h on heart infusion plates containing 0, 64, 256 and 1024 mg/L mupirocin, respectively.



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Figure 1. (a) SDS–PAGE of the flagellin protein of P. aeruginosa grown on heart infusion plates containing various concentrations of mupirocin for 16 h. Lane M, molecular weight marker; lane 1, 0 mg/L; lane 2, 64 mg/L; lane 3, 256 mg/L; lane 4, 1024 mg/L. (b) SDS–PAGE of the flagellin protein of P. mirabilis grown on heart infusion plates containing various concentrations of mupirocin for 20 h. Lane M, molecular weight marker; lane 1, 0 mg/L; lane 2, 16 mg/L; lane 3, 32 mg/L; lane 4, 64 mg/L. Numbers on the left are molecular weight markers (kDa). Arrows indicate flagellin bands. Scanning electron micrographs of P. aeruginosa PAO-1 exposed for 16 h to mupirocin at (c) 0 mg/L, (d) 64 mg/L, (e) 256 mg/L and (f) 1024 mg/L; and P. mirabilis HU2001-309 exposed for 20 h to mupirocin at (g) 0 mg/L, (h) 16 mg/L, (i) 32 mg/L and (j) 64 mg/L. Bar, 3 µm.

 
Effect of mupirocin on flagella formation and motility in P. mirabilis

The effect of 16, 32 and 64 mg/L mupirocin on flagella formation was examined in P. mirabilis (Figure 1g–j). After 16 h of exposure to mupirocin, flagella formation was moderate at 16, 32 and 64 mg/L mupirocin (data not shown). After 20 h of exposure, flagella formation was low at 16, 32 and 64 mg/L mupirocin (Figure 1g–j). As for P. aeruginosa, SDS–PAGE of the flagellin protein from P. mirabilis showed a decrease paralleling reduced flagella formation observed by SEM (Figure 1b). The quantitative estimates of the flagellin protein concentrations by the National Institutes of Health Image program were 81.65, 66.15, 66.92 and 60.32 for P. mirabilis grown for 20 h on heart infusion plates containing 0, 16, 32 and 64 mg/L mupirocin, respectively. By motility assay in P. mirabilis, mupirocin at subinhibitory concentrations suppressed bacterial motility in a dose-dependent manner (Figure 2).



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Figure 2. Effect of mupirocin on bacterial motility (expressed as diameter of zone of growth, mm) in P. mirabilis HU2001-309. P. mirabilis was grown on plates with increasing but subinhibitory concentrations of mupirocin for 18 h at 30°C. Results are expressed as mean ± S.D. (error bars) for three experiments.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the present study, we morphologically and biologically evaluated the effect of mupirocin on flagella formation in P. aeruginosa and on motility and flagella formation in P. mirabilis. Previous studies have shown that expression of virulence factors was suppressed by certain antibiotics such as macrolides and fluoroquinolones.3,5,7,12,13 Some macrolides have been reported to inhibit biofilm formation by P. aeruginosa even below the MIC.3,5,7 Our results indicated that mupirocin below the MIC can reduce expression of virulence factors in P. aeruginosa and P. mirabilis. The concentration examined should be clinically achievable, since the concentration of mupirocin applied topically as an ointment at intranasal sites is typically as high as 20 000 mg/L.14,15

We showed that subinhibitory concentrations of mupirocin reduced flagella formation in P. aeruginosa and P. mirabilis. Previous reports have shown that subinhibitory concentrations of macrolides suppressed expression of flagellin dose-dependently in P. aeruginosa and P. mirabilis.5,16 It has been suggested that flagella can have a role in the initiation of biofilm formation.17 Subinhibitory concentrations of mupirocin dose-dependently suppressed bacterial motility in P. mirabilis while reducing flagella formation and flagellin expression. These results indicate that mupirocin inhibits motility by inhibition of the production of flagellin in P. mirabilis.

Mupirocin (pseudomonic acid A) derived from Pseudomonas fluorescens, inhibits bacterial growth by binding isoleucyl tRNA-synthetase encoded by the ileS gene and is mainly used as a topical agent to reduce spread of methicillin-resistant Staphylococcus aureus (MRSA). The las cell-to-cell signalling system (i.e. the ‘quorum-sensing’ system) has been shown to participate in the differentiation of P. aeruginosa biofilms.18,19 The effect of mupirocin consisting of inhibiting expression of virulence factors may affect Pseudomonas biofilm formation via the quorum-sensing system but is unlikely to affect staphylococcal biofilm formation.

Low concentrations of mupirocin may contribute to mupirocin resistance in MRSA.20 Thus, the use of subinhibitory concentrations of mupirocin could be detrimental, in view of the fact that plasmids can transfer mupirocin resistance among staphylococcus strains and that mupirocin resistance in staphylococci, usually mediated by a conjugative plasmid-associated ileS-2 gene, encoding an additional isoleucyl t-RNA-synthetase, is increasing.2124 The possibility of the appearance of mupirocin plasmid-mediated resistance in the Pseudomonas and Proteus species remains unknown after treatment with subinhibitory concentrations of mupirocin but spread of mupirocin-resistant Staphylococcus strains has been shown in previous studies.2024

In conclusion, we have shown that subinhibitory concentrations of mupirocin can reduce flagellin expression and flagella formation in P. aeruginosa. In P. mirabilis, subinhibitory concentrations of mupirocin dose-dependently suppressed bacterial motility, flagellin expression and flagella formation. Thus, mupirocin showed important biological effects against P. aeruginosa and P. mirabilis at subinhibitory concentrations. Although further studies will be required, topical mupirocin may be useful in inhibiting initiation of biofilm formation in these pathogens.


    Acknowledgements
 
This study was supported by a Grant-in-Aid for Scientific Research (13771452) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and a grant for scientific research from the Uehara Memorial Foundation, Tokyo, Japan.


    Footnotes
 
* Corresponding author. Tel: +81-53-435-2788; Fax: +81-53-435-2794; E-mail: horiihm{at}hama-med.ac.jp Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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