Vancomycin-resistant Enterococcus faecalis pose an emerging health risk, but little is known about the precise epidemiology for vancomycin resistance. The glycopeptide resistant was studied using different techniques such as broth macrodilution, agar dilution combined with agar diffusion, morphology cell changes by scanning electron microscopy. Eight VREF isolated from different clinical samples were used. Results showed low level and high level resistant to vancomycin antibiotic at concentration of 64 to 128 μg/ml, but antibacterial activity was reduced to 256 μg/ml, the SEM revaled increased in the cell size with the antibiotic compared to control and standard culture. The technique constitutes simple method for the detection of organism.
Enterococcus spp. are natural inhabitants of the gastrointestinal tract of humans and animals [1,2] but can be also found in soil, water, and vegetables [
Prior to 1990s, enterococci also have been recognized as an important cause of bacterial endocarditis for almost a century [
The acquisition of high level aminoglycoside resistance (HLAR) and vancomycin resistance has limited the therapeutic options available for clinicians. The transfer potential of vancomycin resistant genes from Enterococci to S. aureus have been reported in clinical settings, increases the importance of findings ways to limit the spread of vancomycin resistant Enterococci (VRE) [
The problem of nosocomial enterococcal infection is compounded by emerging antibiotic resistance. The resistance alone does not explain the increase of Enterococci in nosocomial infections, microorganisms can adapt to different organic substances and other forms of environmental stress by several adaptive mechanisms. The exposure of bacteria to sub-MICs (Minimum Inhibition Concentration) of Vancomycin results in a significant alteration of cellular morphology and disturbance of metabolic activity in resistant E. faecalis [
Scanning electron microscopy offers the unique ability to examine surface structures at relatively high resolution and proves particularly useful in the examination of the effect of antibiotics that act on the bacterial cell wall [12- 14]. The present study describes the effect of antibiotic stress on the morphology of vancomycin resistant E. faecalis strains examined by scanning electron microscopy.
The E. faecalis strains used in the present investigation were isolated from clinical samples over six months period from September 2008 and January 2009 from District Govt. hospital and diagnostic centres from Gulbarga region. The strains were isolated from blood, urine, pus and Cerebrospinal fluid sample.
Bacteria were isolated as previously described [
Antimicrobial susceptibility testing was performed on Mueller Hinton agar (Hi-media, India) by the standard disk diffusion method as recommended by the National Committee for Clinical Laboratory Standards [
All strains were screened for vancomycin (Sigma Aldrich Ltd., Bangalore) MICs by the agar dilution method while the disc diffusion method was performed for screening susceptibility to other antimicrobials, by CLSI guidelines [
SEM is used to examine the minor changes in cell morphology of the populations that have adapted to antibiotic stress [
A total of 122 Enterococcus strains were isolated from different clinical samples on bile esculin agar. The species identities of the clinical Enterococccal isolates, includes 76 (62.29%) strains were E. faecalis. The E. faecalis was the predominant isolates from urine, pus, CSF and blood samples.
E. faecalis strains showed resistance to the different antibiotics like vancomycin (77.63%), gentamycin (64.47%) and oxacillin (55.26%) antibiotics, and were found to be multi drug resistant. The isolates were found susceptible to rifamycin (61.84%), teicoplanin (55.26%) streptomycin (52.63%) and tobramycin (51.13%).
All the vancomycin resistant E. faecalis were subjected for vancomycin MIC’s test. Twelve strains showing drug resistance to all the antibiotics tested were selected for the MIC studies. Among them, 8 strains showed MIC in the range of ≥64 μg/ml while other 4 strains exhibited MIC of ≥128 μg/ml. The bactericidal activity was observed at concentration of 256/256 μg/ml and low bactericidal growth at 128/256 μg/ml. The concentration of antibiotic showed bacterial growth to about ten-fold at 24 hrs, with a concentration of 128/256 μg/ml. An increase in 100 fold at 24 hr was observed with a vancomycin concentration of 6/32 μg/ml.
The results of cell morphology of VREF strains examined by scanning electron microscopy (SEM) revealed that in the presence of vancomycin, the cells altered their morphology with respect to different concentrations of the antibiotic. In the absence of vancomycin the cell morphology of control were apparently normal (
Enterococci infections have become increasingly common because of their intrinsic resistance to several antimicrobial agents and their propensity to acquire resistance from the environment [
tance have increased. Determination of glycopeptides activity has a significant role in guiding antibiotic usage. The results of this study confirms that E. faecalis were more resistant to the vancomycin (77.63%), gentamycin (64.47%) and oxacillin (55.26%) and were sensitive to rifamycin (61.84%), teicoplanin (55.26%) and streptomycin (52.63%) The multidrug-resistant Enterococci are being increasingly reported from all over world. Many studies have demonstrated that E. faecium is comparatively mores resistant than E. faecalis. [
Morphological changes of organisms under stressful conditions are the most visible parameters of bacterial adaptation. The changes in morphology as an adaptive response to adverse environmental conditions have already been reported with several bacterial species [25- 27]. In our study the cell morphology of vancomycin treated cells of E. faecalis under SEM provided strong evidence that the presence concentration of Vancomycin is stressful for the bacterial populations, characterized by the large size. The increase in cell size reduces the relative contact surface and consequently reduces the attachable surface for organic (antibiotic) compounds. Therefore, bigger cells can tolerate the stress conditions better than normal cells of the same species.
Our study reveals that bacteria have evolved an adaptive response to the antibiotic stress and have developed drug resistance. This would be an alarming situation as Vancomycin is one of the few drugs used to treat patients with Enterococcus infection.