Detection of Vancomycin Resistance among Clinical isolates of Enterococci
Ruksana Sheik1, Dr. Gopinath P2
1BDS 2nd year, Saveetha Dental College, Saveetha University, Chennai.
2 Senior Lecturer, Department of Microbiology, Saveetha Dental College, Saveetha University, Chennai.
*Corresponding Author E-mail: ruksana.sheik@gmail.com
ABSTRACT:
Vancomycin -resistant Enterococci (VRE), are the bacterial strains of the genus Enterococcus that are resistant to vancomycin. Enterococci bacteria live in our intestines and on our skin, usually without causing problems. But if they become resistant to antibiotics they can cause serious infections, especially in people who are ill or weak. This study is aims to detect the vancomycin resistance in 20 clinical isolates of Enterococcus spp. We found varied resistance pattern by disc diffusion method and none of the isolates was found to be vancomycin resistance. As Vancomycin Resistant Enterococci (VRE) is emerging to be a threat in nosocomial settings, it is very important to check the same for prompt and accurate therapy.
KEYWORDS: Vancomycin Resistant Enterococci (VRE), disc diffusion method, agar screening method.
INTRODUCTION:
Enterococci have transformed over the past century from being an intestinal commensal organism of little clinical importance to becoming the second most widespread nosocomial pathogen and is related with considerable mortality and morbidity.(1,2) The rate of infection and colonization of patients with vancomycin resistant enterococci (VRE) has been increasing significantly and the resistance can be intrinsic or acquired via gene transfer.(1) The occurrence of VRE has vividly increased globally. (2) Extensive usage of vancomycin and extended spectrum cephalosporins in hospitals has likely contributed to the emergence and remarkable increase of VRE over the past 20 years.(3) The National Nosocomial Infection Surveillance (NNIS) system in the USA has reported a significant increase in the percentage of invasive nosocomial Enterococcus strains displaying high-level vancomycin resistance.(4)
Among the different species of Enterococci which have been identified, Enterococcus faecalis, was the most common species associated with the nosocomial infections, followed by Enterococcus faecium, and together they are responsible for 95% of infections caused by Enterococci.(4) Enterococcus faecium and Enterococcus faecalis are of the normal gastrointestinal flora of humans, but they can cause serious infections such as bacterial endocarditis and bacteremia and are often difficult to treat because of inherent resistance to antimicrobial agents.(5,6)There are other species which account for less than 5% of clinical isolates such as E.gallinarum, E.hirae, E.casseliflavus, E.avium, E.durans. (1) Some of the adverse outcomes associated with the infections caused by VRE are extended length of hospital stay, increased cost and increased mortality.(2,4)
This study was mainly undertaken to detect the vancomycin resistance among the different clinical Isolates of Enterococci species. As Enterococci are gaining importance in causing multitude of infections and treatment failure especially in the nosocomial settings in the last few decades, it is very important to study the resistance profile to vancomycin, as it is being considered as last resort to treat such infections produced by this stubborn pathogen.
MATERIALS AND METHODS:
Clinical isolates:
A total of 20 different non-repetitive clinic isolates of Enterococci were collected from different clinical specimens were included in this study. These isolates were identified by standard biochemical parameters as described by elsewhere. Isolates were preserved in semi-solid brain heart infusion medium and stored at 4şC until further use.
Antimicrobial susceptibility test:
Antibiotic susceptibility test was determined for these strains to routinely used antibiotics such as ampicillin (10µ), vancomycin (30µ), teicoplanin (30µ), erythromycin (15µ), ciprofloxacin (5µ), amikacin (200µ), gentamycin (10µ), tetracycline (30µ) and linezolid (30µ) (Hi Media, Mumbai) by kirby-bauer disc diffusion method.(7)
Detection of vancomycin resistance:
Overnight grown cultures of Enterococci species were adjusted to 0.5 Mc Farland standard. 10 µl of culture suspension was spot inoculated on Mueller Hinton Agar containing 6µg/ml of vancomycin. Plates were incubated at 37şC for 24 hours. Strains were found to be resistant if there is a growth in the spot inoculation.(8)
RESULTS:
Sample wise distribution of clinical isolates of Enterococci:
Of the 20 clinical isolates of Enterococci,
12/20 (60%) were obtained from urine, 4/20 (20%) were from blood, 2/20
(10%) and 2/20 (10%) were from stool samples and wound swabs respectively.
Figure 1 depicts sample wise distribution of clinical isolates of Enterococci.
Fig 1: Pie chart showing the sample wise distribution of clinical isolates of Enterococcus spp.
Bacterial isolates:
Out of 20 Enterococci isolates, 14/20 (70%) were found to be E.faecalis, whereas 6/20 (30%) were E. faecium. Figure 2 denotes the species wise distribution of Enterococci from clinical samples.
Antibiotic susceptibility testing
We found increased percentage of isolates were shown to be resistant to all the antibiotics used in this study. For ampicillin, amikacin, erythromycin, gentamicin, our isolates were found to resistant between 80-90%. Better sensitivity was observed in linezolid, teicoplanin and vancomycin antibiotics. The detailed results of antibiotic sensitivity patter of Enterococci was given in table 1.
Table 1: Results of antibiotic sensitivity patter of Enterococci
Antibiotics |
Sensitivity |
Intermediate |
Resistance |
Ampicillin |
1(5%) |
2(10%) |
17(85%) |
Vancomycin |
15(75%) |
1(5%) |
4(20%) |
Teicoplanin |
12(60%) |
3(15%) |
5(25%) |
Erythromycin |
2(10%) |
0 |
18(90%) |
Ciprofloxacin |
6(30%) |
0 |
14(70%) |
Amikacin |
1(5%) |
1(5%) |
18(90%) |
Gentamycin |
2(10%) |
2(10%) |
16(80%) |
Tetracycline |
4(20%) |
4(20%) |
12(60%) |
Linezolid |
18(90%) |
1(5%) |
1(5%) |
Results of vancomycin resistance:
Isolates that showed resistance to vancomycin in disc diffusion method was detected for the same by agar screening method using vancomycin powder. Using this method, all our isolates were found to be uniformly sensitive to vancomycin in a concentration of 6µg/ml, which indicates that there was no Vancomycin Resistant Enterococci (VRE).
Fig 3: Agar screening plates with vancomycin showed uniform sensitivity
DISCUSSION:
Vancomycin
Resistant Enterococci are being reported from different parts of the world with
increasing frequency, although the distribution of such isolates varies widely
in different areas.(9)The percentage of Enterococci with vancomycin resistance
have been increased from 0.3% in 1989 to 11% in 1996.(10) In this study we did
not get even a single strain with vancomycin resistance.
Detection of vancomycin resistance is very
tidious in clinical microbiology setting. Disc diffusion method by placing 30
microgram vancomycin in disc that frequently misidentify the actual status of
vancomycin resistance. MIC determination by broth or agar dilution by E test
method are considered to be the gold standard for determining vancomycin
resistant status. However these methods are not adapted for the routine use in
clinical diagnostic techniques. (11)
CONCLUSION:
In this study we did not find any Vancomycin
Resistant Enterococci. By disc diffusion method, it showed lesser percentage of
vancomycin resistance. However these statins were confirmed to be vancomycin
sensitive strains. This indicates the promptness of agar screening method with
vancomycin. Hence it can be concluded that this method may be included in the
routine drug susceptibility pattern of Enterococci for the better treatment
modalities.
ACKNOWLEDGMENT:
We thank Dr. Kalyani, Professor and Head of the Department of Microbiology, Saveetha Medical College, Chennai for kindly providing the clinical isolates to carry out our research work
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Received on 13.06.2016 Modified on 22.06.2016
Accepted on 08.09.2016 © RJPT All right reserved
Research J. Pharm. and Tech 2016; 9(12):2106-2108.
DOI: 10.5958/0974-360X.2016.00428.5