ULTRASTRUCTURAL ANALYSIS OF ORGANOMETALIC ANTIMICROBIAL COMPOUND AGAINST MULTIDRUG-RESISTANCE CLINICAL ISOLATES

The emergence of bacterial antibiotic resistance is a serious concern in human and animal health. Methicillin-resistant Staphylococcus aureus (MRSA) and Klebsiella pneumoniae carbapenemase producer (KPC) are the persister bacteria and was evolved after decades of antibiotic misuse. In this context, organometallic antimicrobial compounds (OACs) of natural source may play a main role in a next generation of compounds against bacterial infections. The aim of this study was to characterize the ultrastructural effects caused by OAC in MRSA and KPC clinical isolates. In addition to the standard in vitro microbial techniques, we used transmission electron microscopy (TEM) to study the disrupted cell architecture under antibacterial regimen and we correlate this with scanning electron microscopy (SEM) to compare results of both techniques. The OAC was produced in submerged culture by Pseudomonas aeruginosa strain LV, purified by liquid absorption chromatography and the bioactivity was observed for SEM and TEM. To determine the ultrastructural effects of OAC on bacterial cell, 1 mL of culture (1010 CFU) of each MRSA and kpn-KPC strains were incubated in the presence or absence of OAC and spotted on polylysine-coated glass slides and stored at 28 °C for 1 h to dry (SEM) or centrifuged at 4000 rpm/5 min (TEM). The samples were fixed with 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7) for 12 h and processed for SEM or TEM for bacterial analysis. SEM showed that kpn-KPC and MRSA incubated with OAC for one hour, reduced the number of CFU and decrease extracellular matrix formation when compared with. The OAC caused pronounced morphological alterations in the planktonic cells and the cell morphology was completely altered with a disruption of cell wall forming depressions in the bacterial skeleton. Also, OAC, caused cytoplasmic alteration and vacuolization when was analysed by TEM. The plasma membrane was intact and low electron density was observed probably for failure in the ionic pumps of plasma membrane. The bactericidal activity of OAC observed, by SEM and TEM help us to demonstrate that OAC induced cell lysis and degrading the cellular matrix suggesting the high potential of OAC to use as antibacterial agent.