Prevalence and comparison of high-level aminoglycoside resistance in vancomycin-sensitive and vancomycin resistant enterococcus at a tertiary care hospital in Rohillkhand region

Verma D1, Sinha S2, Ved Prakash3

1Dr Deepika verma, 2Dr Shivani sinha, 3Dr Ved Prakash

Abstract

Introduction: Enterococci, originally commensals of gastrointestinal tract, have now become important nosocomial pathogens. The serious infections caused by Enterococci were empirically treated with bactericidal combination of beta lactam drugs and aminoglycoside neither of which are effective alone [1]. But the presence of high level aminoglycoside resistance (HLAR) makes this treatment ineffective. The emergence of VRE (Vancomycin Resistant Enterococcus) strains also resistant to ampicillin and HLAR has made the treatment further difficult. The spread of such strains in a health care environment has to be constrained and so the present study was carried out to highlight the occurrence of HLAR strains and VRE strains in our hospital .The frequency of association of HLAR with VSE (Vancomycin sensitive Enterococci) and VRE (Vancomycin resistant Enterococci) and any significant difference between the two was also studied. Methods: A total of 100 Enterococci were isolated from 6272 clinical specimen during a study period of 1 year. These strains were speciated on the basis of biochemical tests. Antimicrobial susceptibility test was performed by Kirby baeur disc diffusion method. High level Aminoglycoside resistance was tested by two methods 1) 150μg gentamicin and 200 μg streptomycin disc 2) Agar screening method. Similarly, strains resistant to Vancomycin by disc diffusion method were subjected to confirmatory test by determining their MIC by Agar dilution. Results: A total of 100 Enterococci were isolated during a study period of one year. The prevalence of HLAR was found to be 49%.The prevalence of VRE was found to be 12%.VRE strains also possessing resistance to High strength Gentamycin and or streptomycin was 8%.The frequency of association of HLAR in VRE was 1.5 times higher as compared to VSE. Conclusion: The prevalence of High level gentamicin and high level streptomycin resistance amongst Enterococcal isolates in our institute was high. More enterococcal strains were found to be resistant to both gentamicin and streptomycin than to gentamicin or streptomycin alone. The HLAR rate in VRE was 1.5 times higher than in VSE.

Key words: high level aminoglycoside resistance, Vancomycin sensitive Enterococci, Vancomycin Resistant Enterococcus


Manuscript received: 13th Oct 2015, Reviewed: 20th Oct 2015
Author Corrected: 7th Nov 2015, Accepted for Publication: 15th Nov 2015

Introduction

Enterococci , although are commensals of gastrointestinal tract, they have emerged as important nosocomial pathogen in the last decade. Their emergence in past two decade is due to their resistance to commonly used antibiotics namely Aminoglycosides, cephalosporins, aztreonam and semisynthetic penicillin.

Enterococci are also known to acquire and transfer resistant genes easily which is a responsible for emergence of HLAR strains, VRE strains, and beta lactamase production [1].

Enterococci are intrinsically resistant to aminoglycoside antibiotic which do not cross the cell wall efficiently. The addition of β-lactam antibiotic however sufficiently disorganizes the cell wall and then aminoglycosides gain access to the ribosomal target [2]. But Enterococci have responded by developing several mechanisms for resistance to the synergistic combination e g. binding protein alterations and beta lactamase production for the penicillin and high level resistance to aminoglycoside by enzymatic degradation or ribosomal alteration leading to loss of synergism [3].

Enterococci resistant to vancomycin have emerged as a significant problem in the healthcare system. Glycopeptide antibiotics exert their effect by inhibiting bacterial cell-wall synthesis; VRE strains on the other hand manufacture cell-wall precursors with decreased affinity for the glycopeptide, which prevents the antibiotic from blocking cell-wall synthesis [1]. Enterococci strains which are resistant to multiple antibiotics including ampicillin and aminoglycosides (including high-level resistance), are frequently responsible for therapeutic problems.
    
This study was designed to study the prevalence of VRE and HLAR strains of Enterococci, and whether there was any correlation between HLAR and Vancomycin-Sensitive Enterococci (VSE) and Vancomycin-Resistant Enterococci (VRE).

Material and Methods

100 Enterococcal strains were isolated during a study period of one year from December 2012 to December 2013 from Rohillkhand Medical College and Hospital, a tertiary care hospital of northern India in the state of  Uttar Pradesh.

Gram staining, catalase reaction, bile aesculin, growth in 6.5 per cent NaCl and Sugar fermentation tests were performed to identify and confirm Enterococci. Antimicrobial susceptibility test was performed by Kirby baeur disc diffusion method. [4]

High level Aminoglycoside resistance was detected by following methods:

Two methods were used for detection of HLAR strains

1. High content disc: Disc of 120 μg gentamicin and 300 μg of streptomycin were used for disc diffusion testing. Interpretation was as follows: A zone of less than or equal to 6 mm = Resistant;7–9 mm = Inconclusive; ≥ 10 mm = Susceptible. Resistant: is not synergistic with cell wall–active agent (eg, ampicillin, penicillin, and vancomycin). Susceptible: is synergistic with cell wall–active agent (eg, ampicillin, penicillin, and vancomycin). If disk diffusion result was inconclusive an agar screen test was performed.

2. Agar screen Method: Agar screen method was used to detect high level aminoglycoside resistance amongst the Enterococcal isolates. Brain heart infusion Agar containing 500 g of gentamycin and 1000 g of Streptomycin were prepared. The plates were divided into small sectors and on each sector standard innoculum of each bacterial isolate was innocculated. 5-6 colonies of the isolate were suspended in brain heart infusion broth and concentration was adjusted to 0.5 McFarland standard (1.5×108 cfu/ml). 10 μl of the suspension was spot inoculated on to Brain heart infusion Agar containing gentamycin 500μg/ml and streptomycin 1000μg/ml. The plates were incubated at 37⁰C for 24-48 hrs. Any growth on the plate indicated resistant strain of Enterococci means no synergism with cell wall active agent.

Vancomycin resistance: Strains resistant to vancomycin disc (30μg) were screened for Vancomycin resistance by using Vancomycin screen agar (VSA) containing Vancomycin at concentration of 6 μg/ml. Growth of >1 colony was interpreted as Resistant. Confirmation of VRE was done by determining their MIC by Agar dilution method. Control strains used were E.faecalis ATCC-29212(susceptible) and E.faecalis ATCC-51299(resistant). MIC interpretative standards according to CLSI guidelines is susceptible ≤ 4, 8–16 intermediate and ≥ 32 resistant or VRE. [5]

Results were evaluated according to the Clinical laboratory Standards Institute (2011) [5] and were analysed statistically using the 2 test.

Observation and Result

100 Enterococcus strains were isolated during a study period of one year from 6572 different clinical specimen. Of these 56 strains were isolated from urine, 33 from pus, 04 from vaginal swabs, 05 from blood cultures and 02 from body fluids (ascitic fluid). No Enterococci were isolated from catheter tip, CSF and pleural fluid.

75 isolates were identified as E. faecalis , 20 isolates as E. faecium, 2 were identified as E. avium, 1 each of E.dispar, E. durans and E. raffinosus by biochemical tests .

Resistance to the High Strength Gentamicin 120μg disc were shown by 21 isolates , 05 were inconclusive or intermediate sensitive and 74 were sensitive. Similarly, out of 100 isolates, 34 were resistant to High Strength streptomycin 200μg disc, 62 were sensitive and 04 isolates remained inconclusive.

Out of 75 isolated E. faecalis species, 15 were resistant to high-level gentamicin and 24 were resistant to high-level streptomycin by disc diffusion method.

Table-1: High Level Aminoglycosides Resistance (HLAR) Amongst Enterococci Isolate

Species

Total No. of Isolates

Resistant to Gentamicin

Resistant to Streptomycin

Disk

Diffusion

Screen Agar

Disk

Diffusion

Screen Agar

E. faecalis

75

15

17

24

26

E. faecium

20

4

6

8

8

Other spp.

5

2

2

2

2

Total

100

21

25

34

36

Table-2: High-level aminoglycoside resistance rate of Vancomycin-sensitive Enterococci and Vancomycin-resistant Enterococci using standard agar screening

 

Vancomycin Resistant Enterococci(n=12)

Vancomycin Sensitive Enterococci(n=88)

P value

High level gentamycin resistance(HLGR)

6/12(50%)

19/88(21.5%)

< .01

High level Streptomycin resistance(HLSR)

8/12(66.6%)

28/88(31.8%)

<.01

High level Amino glycoside resistance(HLGR+HLSR)

8/12(66.6%)

41/88(46.4%)

>.05

Table-3: Distribution of HLAR among vancomycin resistant and vancomycin sensitive Enterococcal strains

Enterococcal strains

  HLSR

  HLGR

VRE Faecalis(n=6)

4

3

VRE faecium(n=4)

3

2

VRE avium(n=1)

1

1

VRE durans

(n=1)

0

0

Vre dispar(n=0)

0

0

VRE raffinosus(n=0)

0

0

VSE faecalis(n=69)

22

14

VSE faecium(n=16)

4

4

VSE dispar(n=1)

0

0

VSE raffinosus(n=1)

1

0

Of the 20 E. faecium isolates, 04 were resistant to high-level gentamicin and 08 were resistant to high-level Streptomycin by disc diffusion method. (table 1)

Of the other 05 isolates including E. avium, E. dispar, E. durans and E.raffinosus species, high-level resistance to gentamycin was shown by 02 isolates and high-level streptomycin resistance by 02 isolates. Enterococcus isolates which were resistant to one aminoglycoside, not necessarily be resistant to another aminoglycosides.

In the detection of HLAR using high potency disc all the intermediate sensitive or inconclusive strains when tested by HLAR screen agar, the number of HSG resistant isolates changed to 25 and HSS resistant isolates to 36.

In total 49% of Enterococci showed HLAR in our set up out of which 04% were resistant to gentamicin only, 14% isolates were resistant to streptomycin only and 31 isolates (31%) were resistant to both antibiotics.

But, there were no significant differences between the two methods used to determine the aminoglycoside resistance rates in the enterococcal isolates.

Of the 100 isolates, 12 (12%) were resistant to vancomycin with MIC≥ 32μg/ml.
    
Although High-level aminoglycoside resistance was 1.5 times more in VRE isolates than HLAR in VSE isolates ,this difference was statistically not significant.(p value> .05)

High-level aminoglycoside resistance to both streptomycin and gentamicin was more common in E. faecium than in E. faecalis strains

Discussion

With the emergence of high level amino glycosides resistance (HLAR), β-lactamase producing Enterococci and glycopeptides resistance including Vancomycin Resistant Enterococci (VRE), a study about their spectrum of infections and antibiotic sensitivity pattern becomes important. The difficulty of treating infections caused by Vancomycin resistant Enterococci (VRE) strains which might be resistant to all antimicrobial agents used for the treatment of systemic infections, also emphasizes the need of detection of such strains accurately .[6]

High levels aminoglycoside resistant Enterococci were first reported in France in 1979 and since then have been isolated from all the continents. High level aminoglycoside resistant Enterococci often have plasmids which carry determinants encoding resistance to other antibiotics, besides limiting the option of using a combination of cell wall active antibiotics and aminoglycosides. [7]

The emergence of vancomycin resistance Enterococci in addition to the increasing incidence of high-level enterococcal resistance to penicillin and aminoglycosides presents a serious challenge for physicians treating patients with infections due to these micro-organisms.

The prevalence of HLAR was high in our set up constituted to be 49% while the prevalence of VRE was 12%. Latika shah in 2012 has also reported high HLAR 53% for gentamycin and 40 % for streptomycin with 8% VRE at their institute [8]. Nearly 50 per cent of the isolates showing high level aminoglycoside resistance (HLAR) and a total of 13 (8.6%) isolates showing vancomycin resistance have also been reported by Sanal C Fernandes and B Dhanashree in 2012 [9] Luna adhikari in 2010 [10]and E. padmasini et al [11] in 2013 have also reported high prevalence of HLAR at their setting. Thus a high prevalence of HLAR along with increasing VRE has been reported from all parts of India. Such strains may disseminate in a health care setting and therefore routine screening of all enterococcal isolates with High strength Gentamycin is important.

In the present study, VRE strains also resistant to high level gentamycin or streptomycin was 8%. The clinical importance of HLAR in VRE isolates, however, is much greater than in other strains because of the lack of synergy among glycopeptide antibiotics and aminoglycosides.

Also, HLAR was found to be one and a half (1.5 times) higher in VRE isolates than in isolates of VSE (66.6% in VRE and 46.4% in VSE. When statistically analysed this association was however clinically not significant.(p value>.05)

Further more, such resistance in association with resistance to other antimicrobial drugs may spread to other Gram positive bacteria including Staphylococcus aureus, which may theoretically result in groups of organisms for which there is no effective antimicrobial treatment. Considering this possible risk, all clinically significant isolates of Enterococci should be examined for their antibiotic sensitivity pattern, including HLAR, before the administration of a β-lactam or glycopeptide antibiotic in combination with an aminoglycoside.

Also, in the present study, Linezolid was found to be 100% sensitive in all Enterococcal isolates by Kirby baeur disk diffusion method of antibiotic sensitivity testing. As, Linezolid is also available in oral form and rapidly completely absorbed after oral administration with a mean bioavailability of approximately 100%, Linezolid can be safely used in severe Enterococcal infection and seems to be an appropriate therapeutic option [12].

Conclusion

The prevalence of High level gentamicin and high level streptomycin resistance amongst Enterococcal isolates in our institute was high. More enterococcal strains were found to be resistant to both gentamicin and streptomycin than to gentamicin or streptomycin alone. The HLAR rate in VRE was one and a half times higher than in VSE.    

Funding: Nil, Conflict of interest: None initiated.
Permission from IRB: Yes

References 

1. Randhawa VS1, Kapoor L, Singh V, Mehta G. Aminoglycoside resistance in enterococci isolated from paediatric septicaemia in a tertiary care hospital in north India. Indian J Med Res. 2004 May;119 Suppl:77-9.

2. Marothi YA, Agnihotri H, Dubey D. Enterococcal resistance--an overview. Indian J Med Microbiol. 2005 Oct;23(4):214-9. [PubMed]

3. Aleksandrowicz I. [The incidence of high-level aminoglicoside and high-level beta-lactam resistance among enterococcal strains of various origin]. Med Dosw Mikrobiol. 2012;64(1):11-8.

4. Koneman E.W ,Allen S.D ,Janda W.M,Schreckenberger P.C,WinnW.C, chapter 13. In: Koneman ‘sColour Atlas and Text Book of Diagnostic Microbiology,6 th ed; Baltimore Lippincott William and Wilkins 2006 :726-733.

5. CLSI performance standards for antimicrobial susceptibility testing: 21st information supplement, CLSI M100-S 21 volume 31 no l. No. 1 Fred C. Tenover. CLSI 2011. [PubMed]

6. Sood S1, Malhotra M, Das BK, Kapil A. Enterococcal infections & antimicrobial resistance. Indian J Med Res. 2008 Aug;128(2):111-21. [PubMed]

7. Yazgi H , Ertek M, Erol S and Ayyildiz A. A  Comparison of High-level Aminoglycoside Resistance in Vancomycin-sensitive and Vancomycin resistant Enterococcus Species. The Journal of International Medical Research 2002; 30: 529 – 534.

8. Shah L, Mulla S, Patel KG, Rewadiwala S. Prevalence of enterococci with higher resistance level in a tertiary care hospital: a matter of concern . Natl J Med Res. 2012; 2(1): 25-27.

9. Fernandes SC, Dhanashree B. Drug resistance & virulence determinants in clinical isolates of Enterococcus species. Indian J Med Res. 2013 May;137(5):981-5. [PubMed]

10. Adhikari L. High-level Aminoglycoside Resistance and Reduced Susceptibility to Vancomycin in Nosocomial Enterococci. J Glob Infect Dis. 2010 Sep;2(3):231-5. doi: 10.4103/0974-777X.68534.

11. Padmasini E, Padmaraj R, and Srivani R S. High Level Aminoglycoside Resistance and  Distribution of       Aminoglycoside Resistant Genes among Clinical Isolates of Enterococcus Species in Chennai, India     .Hindawi Publishing Corporation. Scientific World Journal Volume 2014, Article ID 329157, 5 pages. http://dx.doi.org/10.1155/2014/329157.

12. Howden P Benjamin, Ward B Peter, Charles GP Patrick, Korman M Tony et al. Treatment Outcomes for Serious Infections Caused by Methicillin-Resistant Staphylococcus aureus with Reduced Vancomycin Susceptibility. Oxford Journals, Medicine, Clinical Infectious Diseases. 38( 4): 521-8.


How to cite this article?

Verma D, Sinha S, Ved Prakash. Prevalence and comparison of high-level aminoglycoside resistance in vancomycinsensitive and vancomycin resistant Enterococcus at a tertiary care hospital in Rohillkhand region. Int J Med Res Rev 2015;3(10):1162-1166. doi: 10.17511/ijmrr.2015.i10.210.