INTRODUCTION:

Enterococci has been considered as an important nosocomial pathogen since 1990s. Enterococci have gained much importance not only being a nosocomial pathogen and also due to its increasingly documented antibiotic resistance. Serious enterococcal infections are often related to treatment and high mortality rate^.[1] Infections by enterococci have traditionally been treated with cell wall active agents such as penicillin or ampicillin in combination with an aminoglycoside (streptomycin / gentamicin), however emergence of high level resistance to aminoglycosides (HLAR), β lactam antibiotics and to vancomycin by some strains, together with association of HLAR with multi drug resistance has led to failure of synergistic effects of combination therapy.^.[1,2]

Streptomycin was the aminoglycoside used clinically until 1970 when more than 50% of enterococci were appeared to be resistant to high level of this drug.^[1]High level gentamicin resistance (HLGR) was first reported in E.faecalis in 1979. Based on this background, we have undertaken this study to detect the high level aminoglycoside resistance among the clinical isolates of Enterococcus spp.

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 4ºC 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.^[3]

Detection of HLAR:

HLAR in enterococci was detected by disk-diffusion method and agar-screening method. In disk-diffusion method, isolated colonies of enterococci were inoculated into peptone water to get bacterial suspension that was equivalent to McFarland 0.5 standard. Lawn culture on blood agar was done by swabbing the bacterial suspension. High-level (120 μg) gentamicin and streptomycin (300 μg) disks were placed on the agar medium. Plates were incubated at 37°C for 24 hours, and diameter of zone of inhibition was measured. Resistance was indicated by no zone; and susceptibility, by a zone of diameter ≥10mm. ^[4]

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.

Fig 2: Pie chart showing species distribution of Enterococcus spp

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 HLAR among clinical isolates of Enterococci:

The present study showed 11/20 (55%) were found to be HLAR Enterococcus spp, in which 9/11 (81.8%) were in E. faecalis and 2/11 (18.1%) in E. faceium.

DISCUSSION:

Recent years have witnessed increased interest in enterococci not only because of their ability to cause serious infections but also because of their increasing resistance to many antimicrobial agents^.[1,5]. The present study evidences 11/20 (55%) were found to be HLAR Enterococcus spp, in which 9/11 (81.8%) were in E. faecalis and 2/11 (18.1%) in E. faceium. Antibiotic resistance in enterococci is either intrinsic or acquired. Intrinsic traits expressed by enterococci include resistance to semisynthetic penicillinase resistant penicillins, cephalosporins, low level of aminoglycosides and low level of clindamycin, whereas acquired resistance includes resistance to chloramphenicol, erythromycin, high level of clindamycin, tetracycline, high level of aminoglycosides, penicillin, fluroquinolones and vancomycin.^[1] HLAR is due to release of various amnoglycoside modifying enzymes.

Study conducted by Mendiratta and coworkers in 2008, reported that 46% of the enterococci showed HLAR and combined HLGR and HLSR was significantly higher in E. faecium (59.1%) than E. faecalis (7.8%),^[6] as also reported by Gordon et al.^[7]High HLGR in E. faecalis and HLSR in E. faecium observed has also been reported ^[8] as also vive a versa^[7]and no such difference.^[9]Not in agreement with his study, we have reported only less percentage of HLAR status isolates.

CONCLUSION:

As Enterococcus species is one of the very important nosocomial pathogen appeared to resist multitude of antibiotics particularly aminoglycosides, it is important to screen for HLAR status for proper outcome of patients.

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.

REFERENCES:

1. Murray BE. The life and times of the Enterococcus. Clin Microbiol Rev.3;1990:46-65.

2. Jesudason MV, Pratima VL, Pandian R, Abigail S. Characterization of penicillin resistant Enterococci. Indian J Med Microbiol.16;1998:8-16.

3. Clinical Laboratory Standards Institution: Performance standards for antimicrobial susceptibility testing. In NCCLS approved standard M2-A8. Wayne, PA USA: CLSI,2015.

4. Swenson JM, Hindler JF, Jorgensen JH. Special phenotypic methods for detecting antibacterial resistance. In: Murray PR, Baron EJ, Jorgensen JH, Pfaller MA, Yolken RH, editors. Washington DC: ASM Press;2003:1178–95.

5. Patterson JE, Zervos M. High-level gentamicin resistance in Enterococcus: Microbiology, genetic basis and epidemiology. Rev Infect Dis.12;1990:644-51.

6. Mendiratta DK, Kaur H, Deotale V et al. Status of high level aminoglycoside resistant enterococcus faecium and enterococcus faecalis in a rural hospital of central india. Indian Journal of Medical Microbiology. 26(4);2008:369-71.

7. Gordon S, Swenson J, Hill BC, Pigott NE, Facklam RR, Cooksey RC, et al. Antimicrobial susceptibility patterns of common and unusual species of Enteroccoci causing infections in United States. J Clin Microbiol.30;1992:2373-8.

8. Bhat KG, Chitra P, Bhat M. High level aminoglycoside resistance in Enterococci isolated from hospitalized patients. Indian J Med Res.105;1997:198-9.

9. Karmarkar MG, Gersham ES, Mehta PR. Enterococcal infections with special reference to phenotypic characterization and drug resistance. Indian J Med Res.119;2004:22-5.

Received on 27.06.2017 Modified on 04.08.2017

Research J. Pharm. and Tech 2017; 10(12): 4195-4197.

DOI: 10.5958/0974-360X.2017.00765.X