1 Centre of Respiratory Pharmacology, Department of Pharmacology, School of Medicine, University of Milan, Via Vanvitelli 32, 20129 Milano; 2 Department of Biophysical Electronic Engineering, University of Genoa, Italy
Received 13 November 2001; returned 27 May 2002; revised 28 June 2002; accepted 17 July 2002
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Abstract |
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Keywords: atomic force microscopy, Streptococcus pyogenes M phenotype, erythromycin A, rokitamycin
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Introduction |
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Two recent multicentre studies in Italy showed that the average phenotype distribution of resistant strains is 60% MLSB (32% constitutive, 28% inducible) and 40% phenotype M (efflux)-mediated resistance.8,9 A further particular aspect is not only the noticeable presence of the constitutive phenotype, but the fact that the incidence of the M phenotype is similarly high, although with striking differences in incidence in adjacent geographical areas.8,10
The mechanisms of macrolide resistance have been widely investigated and researchers have concentrated on developing new 14- and 15-membered ring macrolides in an attempt to overcome the problem. In addition, published data show that 16-membered ring macrolides, such as josamycin, spiramycin, myocamycin and rokitamycin (the most recent semi-synthetic 16-membered ring macrolide introduced in clinical practice), have antibacterial activity even in the presence of some type of resistance to erythromycin A.1113 Investigating the surface structure of bacteria makes it possible to investigate the efficacy (mechanism of action) of antibiotics that disrupt their structure as an epiphenomenon of internal biochemical action (i.e. ß-lactams),1416 or their lack of activity, as in the case of resistant strains.17 Studies of bacterial morphology and structure are generally based on optical or scanning electron microscopy, but new opportunities have arisen with the recent introduction of atomic force microscopy (AFM).18,19 AFM does not use photons or electrons, but a very small sharp-tipped probe located at the free end of a cantilever driven by the interatomic repulsive or attractive forces between the tip and the surface of the specimen.18,19 Although scanning electron microscopy is still used frequently, the introduction of the new AFM technique offers susbtantial benefits: real quantitative data acquisition in three dimensions; minimal sample preparation times; flexibility in ambient operating conditions (i.e. without the need for a vacuum or gold sputtering); and effective magnifications at the sub-micron level.18,19
The aim of this study was to investigate the disrupting morphostructural effects induced by rokitamycin and erythromycin A on M phenotype S. pyogenes by means of AFM, which has the unique capability of providing high resolution, three-dimensional images of surface structures.
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Materials and methods |
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Three recent clinical isolates of S. pyogenes M phenotype (identified by double-disc test) were used. Suspensions of each organism were prepared from overnight cultures in MuellerHinton broth (Oxoid, Milan, Italy) with fetal calf serum at 37°C under static conditions.
Erythromycin A and rokitamycin were obtained from Sigma (Milan, Italy) and Prodotti Formenti (Milan, Italy), respectively. Stock solutions were prepared by dissolving 2 mg in ethanol, and working solutions were diluted to the appropriate concentrations in MuellerHinton broth.
An inoculum of 106 cfu of the organisms was added to MuellerHinton broth containing serial two-fold dilutions of the antibiotics, in order to determine their MICs under the same conditions as those used to culture the bacteria. After incubation at 37°C for 18 h in ambient air, the MIC was recorded as the lowest concentration of antibiotic that completely inhibited visible growth of the organism.
The mean rokitamycin MIC for the S. pyogenes strains was 0.5 mg/L, whereas no inhibition of bacterial growth was observed with up to 32 mg/L erythromycin A. All strains were then grown in medium with or without antibiotics at 37°C under static conditions. At 0, 2, 4 and 6 h, a 1 mL aliquot was removed from the cultures and prepared for AFM investigation.
Preparation of bacterial samples for AFM studies
For each antibiotic concentration and each incubation time, samples of each S. pyogenes strain were collected, washed three times with phosphate-buffered saline and centrifuged. The final pellet was fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.1), and dehydrated in graded alcohol or simply dried in air. None of the samples underwent drying to the critical point or gold sputtering.
AFM
An Autoprobe CP atomic force microscope (Park Scientific Instruments, Sunnyvale, CA, USA) was used for all AFM imaging. The microscope was equipped with a scanner that had a maximum xy scan range of 100 x 100 µm, at a z-range of 7 µm, which was operated by means of a real-time closed loop scanning control system that allows for the accurate measurement, repositioning and zooming in on selected features (ScanMaster; Park Scientific Instruments). The scanner was calibrated in the three directions by means of a VLSI reference standard (VLSI Standard, San José, CA, USA). The images were acquired using silicon cantilevers with high aspect ratio conical silicon tips (Ultralevers; Park Scientific Instruments). The force constants were 0.03 N/m for contact mode imaging and 7.4 N/m for intermittent contact mode imaging. In order to be able to locate the area of interest on the samples and identify any damaged bacteria, we used the built-in long-range on-axis microscope, which is capable of a 5:1 zoom and x3500 magnification. Intermittent contact mode imaging, which makes it possible to use higher scan rates and which can cope more easily with steep features, was used to acquire images of whole bacteria at scan speeds between 5 and 50 µm/s. Contact mode imaging used at scanning speeds between 1 and 10 µm/s gave less satisfying results due to strong adhesion phenomena between tip and sample, giving rise to image blurring. Accurate feedback tuning was necessary in both imaging modes in order to obtain the maximum possible gain that allowed the resolution of bacteria surface structures while avoiding oscillations when scanning along the side walls of the cell. All images were acquired as 512 x 512 pixels, and processed by means of plane-fitting, high-frequency filtering and three-dimensional shaded rendering. Cross-sections of interesting features were obtained by using the image analysis software of the microscope to acquire numerical topographical information. A typical imaging session began by using the built-in optical microscope and moving the xy table in search of bacteria showing signs of damage. The AFM cantilever was then moved toward the surface in the proximity of the chosen bacterium. A large scan (50 x 50 µm) was made in order to assess the exact position and nature of the bacterium, with further smaller scans being used to zoom in on any interesting features.
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Results |
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As streptococci keep dividing along the same plane as in the previous division and do not separate readily after they divide, they assume the shape of a chain. The length of the chain is highly variable, and individual or double cocci may also be present together with chains. An example of the normal morphology of M phenotype of S. pyogenes in the absence of compounds is shown in Figure 1a. Figure 1 (b and c) shows the appearance of S. pyogenes after 2 h of incubation with erythromycin A (32 mg/L) and rokitamycin (2 mg/L)no morphological changes can be seen.
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Discussion |
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Little attention has so far been given to 16-membered ring macrolides, but the fact that they proved to be effective against bacteria bearing the mef genes encoding a membrane protein responsible for efflux-mediated erythromycin A resistance20 makes it worth considering these macrolides as a means of counteracting resistance to the 14- and 15-membered ring macrolides.
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Acknowledgements |
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Footnotes |
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References |
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