Differences in the susceptibility of Streptococcus pyogenes to rokitamycin and erythromycin A revealed by morphostructural atomic force microscopy

Pier Carlo Braga1,* and Davide Ricci2

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The aim of this study was to use atomic force microscopy (AFM), an innovative type of microscopy, to investigate the different behaviours of erythromycin A (a 14-membered ring) and rokitamycin (a 16-membered ring) in disrupting the morphology of Streptococcus pyogenes with the M phenotype. AFM scanning and sensing of the topography of a sample makes it possible to obtain simultaneous high-resolution digital measurements of the x, y and z coordinates at any point on the bacteria surface. The images obtained before and 2, 4 and 6 h after incubation with erythromycin A (32 mg/L) and rokitamycin (2 mg/L) clearly show that not even high concentrations of erythromycin A interfere with the M phenotype of S. pyogenes, whereas rokitamycin has a progressive action that leads to the formation of abnormally large cells, the loosening of chain structure and the formation of clusters.

Keywords: atomic force microscopy, Streptococcus pyogenes M phenotype, erythromycin A, rokitamycin


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Streptococcus pyogenes is one of the most frequently isolated microorganisms found in clinical practice.1 The importance of group A streptococcal infections has increased over recent years in North America and Europe, and in other parts of the world,2 because of the growing percentage of erythromycin A-resistant strains.35 By means of the erythromycin A–clindamycin double-disc test, three macrolide resistance patterns can be recognized in S. pyogenes.6,7 The absence of a significant zone of inhibition around both discs indicates constitutive MLSB resistance (cMLS phenotype), blunting of the clindamycin zone of inhibition proximal to the erythromycin A disc indicates inducible resistance (iMLS phenotype), and susceptibility to clindamycin with no blunting of the zone of inhibition around the clindamycin disc indicates the M phenotype. In S. pyogenes, MLSB resistance can be mediated by two classes of methylase genes, i.e. the conventional erm(AM) [erm(B)] determinant and the recently described erm(TR) determinant. In contrast, the M phenotype has been shown to be mediated by an efflux system [mef(A)].6,7

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Strains and culture conditions

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 Mueller–Hinton 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 Mueller–Hinton broth.

An inoculum of 106 cfu of the organisms was added to Mueller–Hinton 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.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Because the aim of the study was to highlight the possibilities offered by AFM in investigating the effect of macrolides and the differences in behaviour between erythromycin A and rokitamycin in disrupting the morphological structure of S. pyogenes as an epiphenomenon of their internal biochemical action, the results are confined to the images, and no numerical data will be given.

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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Figure 1. (a) Example of the common morphology of M phenotype S. pyogenes without exposure to compounds. After 2 h of incubation with (b) erythromycin A (32 mg/L) or (c) rokitamycin (2 mg/L), no structural or morphological changes appeared. Bar, 1 µm.

 
Between 2 and 6 h after the beginning of the experiment there was no change in the morphology of the samples incubated in the absence of compounds or with erythromycin A (Figure 2a and b), but the samples incubated with rokitamycin showed a clear alteration in the morphology of the cells and the structure of the chains, which lost their linearity and also included cells of abnormal sizes and shapes (Figure 2c). Greater magnification revealed finer details of the damage induced by rokitamycin.



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Figure 2. AFM images after 6 h of incubation of bacteria. (a) No compounds; (b) with erythromycin A (32 mg/L) no damages were present; (c) with rokitamycin A (2 mg/L) a clear disruption of normal morphology can be seen. Bar, 1 µm.

 
In Figure 3a, the streptococci seem to have completely lost their chain shape; the abnormally enlarged cells are separated from each other and at the same time their engravings separate the surface into three or four parts, a probable sign of a disorganized internal septa formation that generates a cluster structure. The same situation can be observed in Figure 3b, in which cells with still coccoidal albeit abnormal shape can be seen together with a lengthened cell that has no apparent surface engraving, probably because of a loss of septal division.



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Figure 3. At a higher magnification, various aspects of the damages induced by rokitamycin on M phenotype S. pyogenes can be seen clearly. Abnormally enlarged cells, loss of chain structure, formation of clusters, flattening of the cells (a–c) and finally ‘ghost’ formation (d) are visible. Bar, 1 µm.

 
Figure 3c shows a further image of the disrupting effect of rokitamycin: flattened cells are accompanied by cells, such as the one in the close-up, that are losing their cytoplasm. The final result of this situation is the formation of ‘ghosts’, as seen in Figure 3d.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The action of erythromycin A and rokitamycin on the morphology and structural disposition of bacterial cells was investigated by analysing the time-course of the effect. The results clearly show that even high concentrations of erythromycin A were unable to interfere with M phenotype S. pyogenes, as no morphostructural alterations were observed after up to 6 h of incubation with the compound. Under the same conditions, rokitamycin (2 mg/L) began to exert a progressive effect after a lag time of 2 h, characterized by the formation of abnormally large cells, loosening of chain structure and the formation of clusters.

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.


    Acknowledgements
 
We thank M. Dal Sasso, C. Bovio and M. Culici for preparation of bacterial samples. We gratefully acknowledge Formenti (Italy) for the kind gift of rokitamycin. This study was partially supported by a grant from MURST (60%).


    Footnotes
 
* Corresponding author. Tel: +39-2-5031-6990; Fax: +39-2-5031-6990; E-mail: piercarlo.braga{at}unimi.it Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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2 . Nakae, M., Murai, T., Kaneko, Y. & Mitsuhashi, S. (1977). Drug resistance in Streptococcus pyogenes isolated in Japan. Antimicrobial Agents and Chemotherapy 12, 427–8.[ISI][Medline]

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6 . Giovannetti, E., Montanari, M. P., Mingoia, M. & Varaldo P. E. (1999). Phenotypes and genotypes of erythromycin-resistant Streptococcus pyogenes strains in Italy and heterogeneity of inducibly resistant strains. Antimicrobial Agents and Chemotherapy 43, 1935–40.[Abstract/Free Full Text]

7 . Seppälä, H., Nissinen, A., Yu, Q. & Huovinen, P. (1993). Three different phenotypes of erythromycin-resistant Streptococcus pyogenes in Finland. Journal of Antimicrobial Chemotherapy 32, 885–91.[ISI][Medline]

8 . Chelossi, E., Platé, M., De Leo, C. & Schito, G. C. (1997). Attività di alcuni antibiotici su Streptococcus pyogenes isolati recentemente in Italia. Giornale Italiano di Microbiologia Medica Odontoiatrica e Clinica 1, 7–21.

9 . Schito, G. C., Debbia, E. A. & Marinelli, S. (1999). Secondo progetto Artemis Italia. 5. Discussione e conclusioni. Giornale Italiano di Microbiologia Medica Odontoiatrica e Clinica 3, QII, 28–41.

10 . Schito, G. C., Pesce, A. & Debbia, E. A. (1999). Razionale microbiologico per l’uso di antibiotici orali nella terapia delle infezioni respiratorie comunitarie alla luce dell’attuale realtà epidemiologica italiana. Giornale Italiano di Microbiologia Medica Odontoiatrica e Clinica 3, Suppl. A, SA35–58.

11 . Leclercq, R. & Courvalin, P. (1993). Mechanisms of resistance to macrolides and functionally related antibiotics. In Macrolides: Chemistry, Pharmacology and Clinical Uses (Bryskier, A. J., Butzler, J. P., Neu, H. C. & Tulkens, P. M., Eds), pp. 115–23. Arnette Blackwell, Oxford, UK.

12 . Fujii, R. (1991). Rokitamycin, a breakthrough in the family of macrolides. Introduction. In Abstracts of the Seventeenth International Congress of Chemotherapy, Session 97, Berlin, Germany, 1991. Abstract 885. Futuramed Verlagsgesellschaft, Munich, Germany.

13 . Endou, K., Matsuoka, M., Raniguchi, H. & Nakajama, Y. (1993). Implication of cohesive binding of a macrolide antibiotic, rokitamycin, to ribosomes from Staphylococcus aureus. Journal of Antibiotics 46, 478–85.[ISI][Medline]

14 . Gemmell, C. G. & Lorian, V. (1996). Effects of low concentrations of antibiotics on bacterial ultrastructure, virulence and susceptibility to immunodefense: clinical significance. In Antibiotics in Laboratory Medicine (Lorian, V., Ed), pp. 397–452. Williams & Wilkins, Baltimore, MD, USA.

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