Speciation and Antimicrobial Susceptibility pattern of Enterococci from a Tertiary Health Care Center in North India.Correspondence Address :
Dr Varsha Gupta, Professor, Deptt. Of Microbiology, Govt. Medical College & Hospital. Email: email@example.com
Introduction: In recent years, Enterococci have become important nosocomial pathogens. Therefore, it is important for a hospital setting to continuously monitor such infections, and to determine their species and antimicrobial susceptibility pattern from time to time. Keeping these objectives in mind, the present study was conducted in our tertiary health care center in North India.
Methods: A total of 100 enterococcal strains isolated from urine and blood samples were speciated as per the scheme of Facklam and Collins. Antibiotic susceptibility was determined for Amoxycillin, Penicillin, Cephalexin, Erythromycin, Cotrimoxazole, Gentamicin, Vancomycin, Teicoplanin, Linezolid, Imipenem, Piperacillin, Ampicillin- sulbactam, and Nitrofurantoin by the Kirby Bauer disc diffusion method. MIC detection was done by the agar dilution method for penicillin and vancomycin. HLAR detection was done by the agar dilution method for gentamicin and streptomycin, by supplementing the Mueller Hinton agar with 500μg/ml and 2000 μg/ml of the antibiotics, respectively.
Results: 96 of the strains were of Enterococcus faecalis, and 4 were of Enterococcus faecium. Antibiotic susceptibility tests showed a high level of resistance to cephalexin (100%), gentamicin(96.42%), cotrimoxazole (87.03%), erythromycin (77.19%), and penicillin (61.17%). However, only two strains were found to be resistant to vancomycin and teicoplanin. All the strains were sensitive to linezolid. HLAR was seen in 75% of the strains for gentamicin, and in 69% strains for streptomycin. In case of penicillin, MIC values were found to be >16 μg/ml for 14 strains (14%). 6 strains had MIC values of upto 4μg/ml for vancomycin. Out of these, one E. faecalis strain turned out to be Vancomycin resistant enterococci (VRE) showing an MIC value as high as 512 μg/ml.
Conclusion: We conclude that enterococcal strains with high rate of resistance to penicillin and aminoglycosides are prevalent in our nosocomial setting, and emergence of the VRE strain has further worsened this situation. There is an urgent need for more rational and restricted use of antimicrobials in order to minimize the selection and spread of such strains.
Since the advent of Vancomycin resistant Enterococci (VRE) by Uttley et al (1) in 1988, Enterococcal infections have been a cause of great concern among the clinicians, especially in nosocomial settings. In western countries, especially in USA, they have been reported as the third most common pathogen associated with blood stream infections, and the second most common isolated pathogen over all (2).
Though primarily, they are opportunistic pathogens, their inherently low virulence is well compensated for, by their intrinsic resistance to antibiotics, and their ability to acquire resistance to several broad spectrum antibiotics (3). Vancomycin resistance in Enterococci not only leaves fewer options for disease management, but also is important due to the potential risk of vancomycin resistant gene transfer from Enterococci to Staphylococcus aureus (4). VRE has been frequently reported from USA and Europe, but there have not been many reports on their isolation from many Asian countries, including India (5). In addition to it, Enterococci also show an acquired high level resistance to Aminoglycosides (HLAR). Traditionally, a combination of penicillin/ampicillin with an aminoglycoside was the treatment of choice for Enterococci, with vancomycin as the last resort. Therefore, VRE along with HLAR has made the treatment of these infections extremely difficult, and they pose a great challenge to health professionals.
Although 12 species in the Genus Enterococcus have been recognized, the most common species implicated in human infection is E. faecalis (causing 90% of the infections), followed by E. faecium. E. faecium predominantly is the more resistant species than E. faecalis, and emergence of vancomycin resistance in it has caused an increase in the frequency of its isolation (6). Considering all these facts, the present study was conducted in the tertiary health care centre of North India, to speciate as well as to study the antibiogram of enterococcal strains isolated from clinical samples â€“urine and blood. Minimum inhibitory concentration (MIC) values were also determined for penicillin and vancomycin, along with HLAR detection in these isolates.
The present study was conducted in the Department of Microbiology, Government Medical College Hospital, and Chandigarh. A total of 100 strains of Enterococci were isolated from clinical samples namely- urine (49) and blood (51). The strains isolated were identified and speciated according to standard laboratory procedures as per the scheme of Facklam and Collins (7).
Antimicrobial susceptibility testing was done by the Kirby- Bauer disc diffusion method as per the recommendations of CLSI (8). Various antibiotics tested were: Amoxycillin (10 μg), Penicillin (10units/disc), Cephalexin (30 μg), Erythromycin (15 μg), Cotrimoxazole (25 μg),Gentamicin (30 μg), Vancomycin (30 μg), Teicoplanin (30 μg), Linezolid (30 μg), Imipenem (10μg), Piperacillin (100 μg), Ampicillin- sulbactam (10/10 μg), and Nitrofurantoin (300 μg).
MIC detection was done by the agar dilution method (9) for penicillin and vancomycin for the MIC values of 2 â€“ 512g/ml. HLAR detection was done by the agar dilution method for gentamicin and streptomycin by supplementing the Mueller Hinton agar with 500 μg/ml and 2000 μg/ml of the antibiotics, respectively.
The source of media and antibiotic discs was Hi - Media Ltd. (Mumbai), India. The standard strain E. faecalis ATCC 29212 was used as control.
Of the total enterococcal strains isolated, 96 of the strains turned out to be those of E. faecalis, and 4 were E. faecium on the final species level identification. We could not isolate any other species of Enterococci from our settings. Antibiotic susceptibility tests showed high level resistance to various antibiotics tested. Only two strains were found to be vancomycin and teicoplanin resistant. All the strains were sensitive to linezolid (Table/Fig 1)HLAR was seen in 75% of the strains for gentamicin and 69% strains for streptomycin(Table/Fig 2). In case of penicillin, MIC values were found to be >16 μg/ml for 14 strains. Out of these 14 strains, 6 had raised MIC values of upto 256 μg/ml. For vancomycin, 6 strains had MIC values upto 4 μg/ml. Out of these, one E. faecalis strain turned out to be Vancomycin resistant Enterococci (VRE) showing an MIC value as high as 512 μg/ml.
Enterococci have turned into significant human pathogens because of a combination of colonizing abilities and drug resistance, both inherent and acquired. In the present study, E. faecalis (96%) was the predominant species isolated, followed by E. faecium (4%). Most of the studies done on Enterococci, support the same finding. The reason could be the predominance of E. faecalis in the endogenous flora of the body (10).
Penicillin, along with aminoglycosides, is the mainstay of therapy for infections with Enterococci. Therefore, resistance of Enterococci against these antibiotics has important clinical implications. In the present study, about 61% of the strains were resistant to penicillin by the disc diffusion method, and 14 (14%) of the strains had raised MIC values (>16μg/ml). 6 of them had MIC values of more than 200 μg/ml, which is considered as cut off for high level resistance to penicillin.2 The mechanism of this resistance could be due to the low affinity penicillin binding proteins, or production of beta lactamases.
Among aminoglycosides, 96% of the isolates exhibited resistance to gentamicin by the disc diffusion method. HLAR was seen in 75% of the strains for gentamicin, and in 69% for streptomycin. HLAR was more in E. faecium than in E. faecalis (Table/Fig 2), as has been reported previously also (11), (12). . Both HLGR and HLSR were seen in 55 isolates. HLAR in these strains can well nullify the efficacy of combination therapy. Therefore, distinguishing HLAR from simple intrinsic resistance is important, and should be adopted as a part of any routine microbiology laboratory.
Only 2 strains were found to be resistant to vancomycin and teicoplanin by the disc diffusion method. Out of these, one strain did not show any rise in the MIC value, but the other strain turned out to be VRE, with a highly raised MIC value with a range of upto 512μg/ml. This strain (VRE) was isolated from the blood sample of a female patient with left Guillain Barreâ€™ Syndrome, with polyneuritis cranialis. Blood samples taken from the patient on days 1 and 3 of admission revealed the growth of Enterococcus species organisms, which on further confirmation, was reported as E. faecalis. On checking by antibiogram, the organism was found resistant to ampicillin, penicillin, erythromycin, gentamicin, cotrimoxazole, imipenem, piperacillin, teicoplanin, and vancomycin, but sensitive to nitrofurantoin and linezolid. MIC detection for vancomycin showed a value of upto 512 μg/ml. The strain also showed positive HLAR for streptomycin, but was negative for HLAR for gentamicin.
The patient was managed conservatively, and was administered a combination drug, piperacillin-tazobactam along with clarithromycin, to which she responded well. In the present case, the organism isolated, probably belongs to the Van A phenotype, as it showed resistance to vancomycin upto an MIC of 512 μg/ml, and was resistant to teicoplanin by the disc diffusion test. The various risk factors associated in this case were, history of previous hospitalization, admisssion to ICU, catheterization, and prolonged antibiotic treatment. Previously from India, there are few reports of emergence of vancomycin resistance in enterococcal strains with increased MIC values (Table/Fig 3) (13), (14), (15), (16). The strain isolated in our case had an MIC value as high as 512 μg/ml. Previously also, only a single case of vancomycin resistant E. faecalis strain with MIC value as high as 512 μg/ml has been reported from the blood sample of an ICU patient from Delhi, India.
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