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Tropical Biomedicine 24(1): 105109 (2007)

Mesosomes are a definite event in antibiotic-treatedStaphylococcus aureus ATCC 25923

Santhana Raj, L.1*, Hing, H.L.2, Baharudin Omar2, Teh Hamidah, Z.1, Aida Suhana, R.1,Nor Asiha, C.P.1, Vimala, B.1, Paramsarvaran, S.1, Sumarni, G.1 and Hanjeet, K.11 Electron Microscopy Unit, Institute for Medical Research, Jalan Pahang, 50588 Kuala Lumpur.2 Department of Biomedical of Science, Jalan Raja Muda Abdul Aziz, 50300, Universiti Kebangsaan Malaysia.E-mail: santhana@imr.gov.myReceived 21 March 2007; received in revised form 11 April 2007; accepted 16 April 2007

Abstract: Mesosomes of Staphylococcus aureus ATCC 25923 treated with antibiotics wereexamined morphologically under the electron microscope. The Transmission ElectronMicroscope Rapid Method was used to eliminate the artifacts due to sample processing.Mesosomes were seen in all the antibiotic treated bacteria and not in the control group. Themain factor that contributes to the formation of mesosomes in the bacteria was the mode ofaction of the antibiotics. The continuous cytoplasmic membrane with infolding (mesosomes)as in the S. aureus ATCC 25923 is therefore confirmed as a definite pattern of membraneorganization in gram positive bacteria assaulted by amikacin, gentamicin, ciprofloxacin,vancomycin and oxacillin antibiotics. Our preliminary results show oxacillin and vancomycintreated bacteria seemed to have deeper and more mesosomes than those treated withamikacin, gentamicin and ciprofloxacin. Further research is needed to ascertain whether thedeep invagination and the number of mesosomes formed is associated with the types ofantibiotic used.

INTRODUCTION

The first electron micrograph of the ultrathin section of a bacterial cell has beendevoted to the elucidation of the finestructure of the bacterial cell. However,the technical difficulties and variant ofspecies of bacteria have limited thescientific world from studying in detail thefine structures of the bacteria, such as theouter membrane, plasma membrane andmesosomes (Hackenberg, 2005). In thisstudy, the focus will be on the mesosomesof the bacteria. Based on theirmorphological and ultra structure aspects,the mesosomes can be classified asmembrane-bounded organelle. Thisstructure could be an extended region ofthe plasma membrane. There is nowgeneral agreement that mesosomes are

present in both gram-positive and gram-negative bacteria (Sidaway, 1980).

The focus of this study is to prove thatthe mesosomes are a real event. Thebacteria will be exposed to five differentkinds of antibiotics. The antibioticsselected are based on their mode of action.The Transmission Electron MicroscopeRapid Method (TEMRM) would be appliedin the processing of the control andantibiotic-treated bacteria groups. Themain idea of using TEMRM is to avoid thepresence of mesosomes due to thepreparation technique of electronmicroscopy. The TEMRM was utilizedbased on the assumption that bacteriabeing a prokaryocyte would be fixed, butthe fixation would not stop the livingprocess of the bacteria.

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MATERIALS AND METHODS

Bacteria cultureS. aureus ATCC 25923 was obtained fromthe Bacteriology Unit, Institute for MedicalResearch, Kuala Lumpur. The concentra-tion of the bacteria used in this study was1.4 X 107 bacteria per test and the ratiobetween the bacteria and the fixativesolution was at 1:15.

Disc diffusion testA suspension of S. aureus ATCC 25923 at1.0 x 108 CFU/ml was made and streakedin at least three directions over the surfaceof the Mueller-Hinton agar to obtain auniform growth. After the plates weredried for five minutes, antibiotic disks ofciprofloxacin, amikacin, oxacillin, genta-micin, and vancomycin were placed on theswabbed agar. The plates were incubatedovernight at 37C. Following incubation,the bacteria around the rim of inhibitiongrowth zone around each disk were usedfor electron microscope analysis.

Transmission electron microscoperapid methodThe bacteria that were fixed for 20 minutesin the 2% glutaraldehyde (GA) in 0.1M PBSwere washed with distilled water (X6).After staining with 2% uranyl acetate (UA)for 5 minutes, the bacteria were exposedto osmium tetraoxide (OT) for 5 minutes.The dehydration of the exposed bacteria toOT was dehydrated with a series ofacetone (50%, 70%, 90% and absoluteacetone) respectively for 5 minutes each.Polymerization was done with pure epoxyresin in an embedding oven at 90C for 2hours after the bacteria have beeninfiltrated by a mixture of acetone andepoxy resin (1: 1) for 15 minutes. Theblocks were trimmed and cut to 90 nmultra thin sections and mounted on 200mesh thin bar copper grids. The specimenswere then stained with Reynolds stain for1 minute. Each specimen was examined at65 000X and 110 000 X magnifications byusing Tecnai G2 TEM at an acceleratingvoltage of 90 KV.

RESULTS AND DISCUSSION

A total of 60 electron micrographs weretaken for analyzing the presence ofmesosomes in the bacteria. Themesosomes were only present in theantibiotics treated bacteria (Figures 1B,1C, 1D, 1E and 1F) and not in the controlgroup (Figure 1A). The vancomycin andoxacillin treated bacteria (Figures 1E and1F) shows a deep invagination ofmesosomes compared to amikacin,gentamicin and ciprofloxacin treatedbacteria (Figures 1B, 1C and 1D). Thenumber of mesosomes was more in thevancomycin and oxacillin treated bacteria(e.g. two or more mesosomes were seen inFigures 1E and 1F) than amikacin,gentamicin and ciprofloxacin treatedbacteria (only one mesosome seen inFigures 1B, 1C and 1D). Moreover,amikacin, gentamicin and ciprofloxacintreated bacteria had much thicker outermembrane than vancomycin and oxacillintreated bacteria (Figures 1B, 1C and 1Dcompared to Figures 1E and 1F).

This study proves that the formation ofmesosomes were indeed a real event,based on the observations of the presenceof mesosomes only in the antibiotic-treated S. aureus ATCC 25923 (Figures 1B,1C, 1D, 1E and 1F). The formation ofmesosomes in bacteria was to act as adefensive mechanism to protect thebacteria from antibiotic assault, therebysustaining the survival of the species. It isalso interesting to note that the plasmamembrane of the bacteria act asmitochondria of eukaryocytes (Pichler etal., 2001). Thorne and Barker (1969)successfully isolated certain enzymes,such as ATPase, acetokinase and phospho-transacetylase from plasma membrane andmesosomes of Lactobacillus casei. Thesesame enzymes can also be found in themitochondria of the eukaryocytes (Pichleret al., 2001). The association between themesosomes and mitochondria was alsosupported by the detection of lipoteichoicacid (the end product of the wallsynthesizing enzymes) in all of these

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Figure 1. The electron micrographs : (A) = control group, (B) = amikacin treated bacteria, (C) =gentamicin treated bacteria, (D) = ciprofloxacin treated bacteria, (E) = oxacillin treated bacteriaand (F) = vancomycin treated bacteria. (E) and (F) electron micrographs show deep invaginationof mesosomes in the bacteria compared to (B), (C) and (D) electron micrographs. (A) electronmicrograph of the control group did not show any mesosomes in the bacteria. M = Mesosomes,PM = Plasma Membrane and OM = Outer membrane. Magnification of (A), (B) and (C) are at 111000X and magnification of (D), (E) and (F) are at 65 000X. Scale bar is at 200 nm.

organelles (Greenawalt & Whiteside,1975).

It was also reported the mitochondriaof the rat liver could be disassembled tosmaller constituent structures, such as the

lamellae and tubules. This is related to theformation of mesosomes in the bacteria.The mitochondria of different species andcell types could react differently dependingon the situation and functions of the

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species and cell types. The harsh condition(the antibiotics environment as in thisstudy) would concentrate the oxidativephosphorylation enzymes and make theplasma membrane of the bacteria morefluid to form the mesosomes. Thesemesosomes would increase the surfacearea to facilitate more efficiently theoxidative reaction (Greenawalt &Whiteside, 1975).

The study also revealed that mem-brane attacking antibiotic (such as thevancomycin and oxacillin) (Figures 1E and1F), caused deep invagination of meso-some in the bacteria compared to the RNAand DNA attacking antibiotics (such asamikacin, gentamicin and ciprofloxacin)(Figures 1B, 1C and 1D). The numbermesosomes present in vancomycin andoxacillin treated bacteria (Figures 1E and1F) were more compared to the amikacin,gentamicin and ciprofloxacin treatedbacteria (Figure 1B, Figure 1C and Figure1D). These could be due to the frequenciesof shutting down the existing channels inthe membrane of the bacteria becomingmore amplified in the vancomycin andoxacillin than the amikacin, gentamicinand ciprofloxacin treated bacteria. Theseamplifications would make the bacteria tobe more active in the plasma membraneenzymes level and allowed the plasmamembrane to be more fluid. The fluidity ofthe plasma membrane would allow themesosomes to perturb deeper in thecytoplasm and increased the number ofmesosomes in the bacteria. We alsonoticed that the amikacin, gentamicin andciprofloxacin treated bacteria (Figures 1B,1C and 1D) had thicker outer membranethan the vancomycin and oxacillin treatedbacteria (Figures 1E and 1F). The thickerouter membranes mesosomes were moreconfined to the plasma membrane than thethinner membranes mesosomes. Theconfinement of the mesosomes to theplasma membrane allowed us to concludethat the thickeing of the outer membraneplayed a role in the fluidity of the plasma

membrane and the formation of themesosomes.

The same event that took place inantibiotics treated bacteria could happenin the bacteria in the fixative solutionsbecause of the initial fixation byglutaraldehyde on the transepithelial Na+

and K+ currents. The research teams ofHiggins and Daneo-Moore (1974) and Silvaet al. (1976) had successfully demonstratedthe formation of mesosomes caused by GAand OT fixations. We believe the durationof fixation they used may have an impacton the formation of the mesosomes. Thus,we modified the fixation time in TEMRMto the generation time of the S. aureus andthereby provided us the true descriptiveanalysis in the formation of mesosomes inthe antibiotics treated conditions.

This study clearly shows that themesosome observed in the antibiotics-treated S. aureus ATCC 25923 was a realevent and the mesosomes could only bepresent if the surroundings were hostile tothe bacterias living condition. In order toavoid any misleading conclusion duringany study of ultra structure analysis ofbacteria, we should choose the rightprotocol of electron microscopy. Con-sideration must be given to generationtime and how the bacteria species react tothe fixative solutions. The TEMRM Methodthat was applied in this study confirmedthat the formations of mesosomes in thebacteria were not due to the TEMpreparation techniques but indeed adefinite event. Further research is neededto ascertain whether the deep invaginationand the number of mesosomes formed isassociated with the types of antibiotic.

Acknowledgement. We like to extend ourgratitude to Dr. Shanaz bin Murad, Directorof Institute for Medical Research and Dr.Amal Nasir bin Mustafa, the Head ofMedical Research Resources Center ofInstitute for Medical Research, Malaysiafor their support in this project.

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