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ORIGINAL ARTICLE  
Year : 2016  |  Volume : 9  |  Issue : 3  |  Page : 165-169
Detection of high-level aminoglycoside resistant pattern of Enterococci isolated from urine samples at a tertiary care hospital in Bengaluru


Department of Microbiology, Vydehi Institute of Medical Sciences and Research Centre, Bengaluru, Karnataka, India

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Date of Web Publication3-May-2016
 

   Abstract 

Aims: Enterococcus species are major nosocomial pathogens and they most commonly cause urinary tract infections (UTIs), exhibiting vancomycin and high-level aminoglycoside resistance (HLAR) with increasing frequency, resulting in high mortality of patients with serious enterococcal infections. Detection of resistance is thus of paramount importance. The present study aims to detect and determine the HLAR pattern of Enterococci isolated from urine samples of patients diagnosed with UTI at our hospital. Materials and Methods: This study was carried out at a tertiary care hospital in Bengaluru for a period of 1 year from January 2013 to December 2013. A total of 105 enterococcal strains were isolated from urine samples and speciated as per the scheme of Facklam and Collins. Antibiotic susceptibility was determined for various drugs by Kirby–Bauer disc diffusion method. The results were interpreted as per the Clinical and Laboratory Standards Institute (CLSI) guidelines. Results: Ninety-three of the 105 (88.6%) isolates showed high-level resistance to gentamicin and/or streptomycin. Combined resistance to both the aminoglycosides, high level gentamicin and streptomycin (HLAR), was seen only in Enterococcus faecalis 20/105 (19.04%). Of the two isolates of Enterococcus faecium, 1 (50%) was seen to be resistant to high level gentamicin. The HLAR E. faecalis and E. faecium isolates also showed concordant resistance to multiple antibiotics including vancomycin. Conclusion: This study highlights the need to screen for HLAR in patients suffering from enterococcal infections. Routine screening for HLAR is important to limit the spread of resistance and to have a surveillance program.

Keywords: Entamoeba dispar, Enterococcus faecalis, Enterococcus faecium, high-level aminoglycoside resistance (HLAR)

How to cite this article:
Huidrom S, Narayanaswamy G, Dadlani R. Detection of high-level aminoglycoside resistant pattern of Enterococci isolated from urine samples at a tertiary care hospital in Bengaluru. Ann Trop Med Public Health 2016;9:165-9

How to cite this URL:
Huidrom S, Narayanaswamy G, Dadlani R. Detection of high-level aminoglycoside resistant pattern of Enterococci isolated from urine samples at a tertiary care hospital in Bengaluru. Ann Trop Med Public Health [serial online] 2016 [cited 2019 Dec 5];9:165-9. Available from: http://www.atmph.org/text.asp?2016/9/3/165/181657

   Introduction Top


Urinary tract infection (UTI) is one of the most common infectious conditions in clinical practice.[1] In the last few years, enterococcal infections have become frequent in hospital settings. Currently Enterococci are an important cause of nosocomial infections with increasingly common isolates that are resistant to multiple antibiotics.[2] UTIs constitute the most common type of disease produced by Enterococcus spp. Enterococcal UTIs are commonly acquired in hospital or long-term care settings, and thus, are more likely to be resistant to many antibiotics. In the intensive care unit (ICU) setting, Enterococci cause almost 15% of health-care-associated UTIs.[2] Not unexpectedly, serious enterococcal infections are often refractory to treatment and the mortality is high.[1],[2]

Infections by Enterococci have traditionally been treated with cell wall active agents (e.g., penicillin or ampicillin) in combination with an aminoglycoside (streptomycin/gentamicin). However, the emergence of high-level resistance to aminoglycosides (HLAR), beta-lactam antibiotics and vancomycin, by some strains, together with multidrug resistance, has led to the failure of synergistic effects of the combination therapy.[2],[3] Streptomycin was the only aminoglycoside used clinically until 1970 when more than 50% of Enterococci sp. were found to be highly resistant to this drug. A study from the Massachusetts General Hospital, Boston, Massachusetts showed that, as early as 1973 to 1976, 54% of strains had HLR to streptomycin.[3],[4]

The present study was undertaken considering the paucity of data on HLAR in Enterococci isolated from urine samples of adult patients suffering from UTIs admitted at a tertiary hospital.


   Materials and Methods Top


Enterococci isolated from all urine samples collected over a period of 1 year, from January 2013 to December 2013, form the diagnostic section of the Department of Microbiology, of our hospital, from the patients, both males and females (>18 years), clinically diagnosed with UTI from various wards identified and speciated by test scheme proposed by Facklam and Collins.[5],[6]

Antibiotic susceptibility testing of the isolated strains of Enterococci was carried out by Kirby–Bauer disc diffusion technique using Mueller Hinton Agar, according to the Clinical and Laboratory Standards Institute (CLSI) guidelines.[7]

The antibiotics tested were penicillin (10 U), ampicillin (10 μg), gentamicin (10 μg), high-level gentamicin (120 μg), high-level streptomycin (300 μg), vancomycin (30 μg), teicoplanin (30 μg), linezolid (30 µg), ciprofloxacin (5 μg), and nitrofurantoin (30 μg).

These antibiotics were obtained as commercial discs from HIMEDIA LABORATORY, Mumbai.

Staphylococcus aureus ATCC 25923 and Enterococcus faecalis ATCC 29212 were used to check quality of routine and high content discs, respectively.


   Results Top


During the study period of 1 year, from January 2013 to December 2013, conducted in a tertiary hospital in Bengaluru. A total of 105 Enterococcus species were isolated from urine samples of the patients clinically diagnosed with UTIs [Table 1].
Table 1: Different Enterococcus species isolated (N = 105)

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Out of the 105 enterococcal isolates, E. faecalis was the predominant species 102 (97.11%). There were two (1.9%) isolates of Enterococcus faecium followed by a single isolate of Entamoeba dispar dispar (0.95%). Sixty-one (58.09%) were female patients with symptoms of UTI and 44 (41.9%) were male patients with symptoms of UTI. Out of the 44 male patients, the maximum age group was found to be 31-40 years age group (29.6%); followed by the 18-30 years age group (25%); and the 61 female patients were seen in the age group of 18-30 years, comprising 44.3% of all cases [Table 2], [Table 3], [Table 4].
Table 2 Distribution of isolates in relation to the patient's sex (N = 105)

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Table 3: Distribution of male patients' age (N = 44)

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Table 4: Distribution of female patients' age (N = 61)

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A total of 93 (88.6%) isolates showed high-level resistance to gentamicin and/or streptomycin by both, high content disc diffusion. HLAR among E. faecalis isolates 92/102 (90.2%) was significantly higher than E. faecium 1/2 (50%) and this was also evident when the two isolates were compared with respect to the two drugs considered individually. Combined resistance to both the aminoglycosides, high-level gentamicin and streptomycin (HLAR), was seen only in E. faecalis 20/105 (19.04%). The single isolate of E. dispar showed no resistance to either of the high-level aminoglycosides [Table 5] and [Table 6].
Table 5: Distribution of high-level aminoglycoside resistance enterococcal species with respect to resistance to aminoglycoside combination

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Table 6: Resistance of HLGR and HLSR E. faecalis and E. faecium to other antibiotics

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Of the two isolates of E. faecium, 1 (50%) was seen to be resistant to high-level gentamicin.

Both E. faecalis and E. faecium showed multidrug resistance. It was observed that the single E. faecium isolate with resistance to HLGR/HLSR also showed resistance to various antibiotics including vancomycin, linezolid, and nitrofurantoin [Table 7].
Table 7: Resistance of HLAR E. faecalis to other antibiotics

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   Discussion Top


Enterococci have emerged as an important nosocomial pathogen in the last few decades and the main reason for this is the trend of increasing antimicrobial resistance seen in these organisms. 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. UTIs are the most common cause of infectious disease produced by Enterococci, both within and outside hospital settings.[1],[2]

In the present study, out of 105 enterococcal isolates from urine samples, only 3 species of Enterococci were isolated. Out of these, E. faecalis was the predominant species [102 (97.14%)], with two isolates of E. faecium (1.9%) and a single isolate of E. dispar (0.9%). The findings were comparable with other studies, such as Shrihari et al.,[8] in which 96% of E. faecalis and one of E. faecium (4%) was isolated from the urine samples. Palanisamy et al.[9] also isolated 91.83% of E. faecalis followed by 7.1% of E. faecium. A study done by Parameswarappa et al.[10] showed 63.3% of E. faecalis followed by 3.67% of E. faecium.

Although in the present study, only two isolates of E. faecium were isolated, it was observed that E. faecium was more multidrug resistantas compared to E. faecalis, showing resistance to penicillin, ampicillin, vancomycin, teicoplanin, linezolid, and high-level gentamicin. It was however not clear whether it was statistically significant as the number of E. faecium isolates was very less. Similar findings have been reported by Telkar et al.[11] and Parameswarappa et al.[10]

A number of strains of Enterococci have acquired HLR (MIC ≥2,000 μg/mL) to an aminoglycoside(s) and, concomitantly, resistance to synergism. A study from the Massachusetts General Hospital, Boston, Massachusetts, showed that, as early as 1973 to 1976, 54% of strains had HLR to streptomycin and 49% had HLR to kanamycin. The prevalence of strains highly resistant to various aminoglycosides varies from place to place but is generally highest for streptomycin. Two mechanisms have been described which account for HLR of clinical isolates to streptomycin: ribosomal resistance and enzymatic modification by an adenylyl transferase.[2],[3]

High-level resistance is usually due to the transferable plasmid-mediated production of aminoglycoside-inactivating enzymes.[3]



HLAR Enterococci were first reported in France in 1979 and since then they have been isolated from all the continents. There is little need to test for aminoglycosides other than streptomycin and gentamicin, as these are the agents with the most clinical data. Till date, all strains with high level resistance (HLR) to gentamicin have also shown resistance to synergism and/or HLR to tobramycin, sisomicin, netilmicin, kanamycin, and amikacin by virtue of the enzyme 2″-APH-6′-ACC. This enzyme is not active against Streptomycin, and thus Gentamicin-resistant strains are not necessarily resistant to streptomycin; in other words, a variable percentage of strains will have HLR to gentamicin while lacking HLR to streptomycin.[2],[3],[10]

Resistance to aminoglycosides is of great concern, since it eliminates the synergy of aminoglycosides with β-lactam antibiotics, which is the therapy of choice for Enterococcal infections, thus limiting the therapeutic options.[10]

In the present study, HLAR was seen in 93 (88.6%) isolates. Of the 102 E. faecalis isolates, 34 (33.3%) showed high level Gentamycin resistance (HLGR), 38 (37.3%) showed high level Streptomycin resistance (HLSR), and 20 (19.6%) showed HLAR (HLGR + HLSR). Telkar et al.[11] in their study also observed HLAR in 49 (55%) and Menidratta et al.[12] observed HLAR in 69 (46%) of isolates, respectively.

Our study showed a high prevalence of HLAR, especially in E. faecalis. The reason for higher prevalence could be because it was conducted at an urban tertiary hospital where chronic cases are prevalent and there is wider usage of broad spectrum antibiotics. Thirty-four (33.3%) of E. faecalis and 50% of E. faecium were resistant to high-level gentamicin. Similar findings were reported by Fernandes et al.[13] (53.5% of E. faecalis and 53% of E. faecium, and in Bhat et al.[14] 48.7% of E. faecalis and 54.6% of E. faecium were resistant to high-level gentamicin.

In our study, 38 (37.3%) of E. faecalis were resistant to high level Streptomycin (HLS). Similar findings were reported by Fernandes et al.[13] 2013 who have reported 48.8% of E. faecalis and 58.8% of E. faecium; 34.1% of E. faecalis and 54.6% of E. faecium as reported by Bhat et al.[14] were resistant to HLS. Resistance to high-level gentamicin was higher in E. faecium than in E. faecalis, similar findings were observed by Gordon et al.[15] who also reported significantly higher resistance to HLG and HLS by E. faecium than in E. faecalis. Similar result was also observed by Mendiratta et al.[12] High HLGR in E. faecalis and HLSR in E. faecium observed has also been reported by Bhat et al.;[14] as also vice versa by Karmarkar et al.[16]

Of all the 20 HLAR isolates of E. faecalis, 14 (70%) also showed resistance to vancomycin by disc diffusion. Only three isolates however showed vancomycin resistance by E-test. One out of two isolates of E. faecium that was resistant by disc diffusion to HLGR and vancomycin was resistant by E-test also.

To conclude, multidrug resistant Enterococci were identified in the study. A very high prevalence of HLAR was noted. Concomitant vancomycin resistance was also seen, as has been reported by thus highlighting the shifting spectrum of antibiotics resistance of Enterococci, especially in an urban setting where antibiotic usage is high. Regular screening of enterococcal isolates from urine specimen for detection of vancomycin and HLAR is thus recommended for the effective treatment of enterococcal UTIs to limit the spread of multidrug-resistant strains.


   Conclusion Top


The problem of HLAR and Vancomycin Resistant Enterococci (VRE) as seen in our study conducted in an urban hospital setting is quite high at present. This study highlights the need to identify enterococcal UTIs and establish a surveillance pattern to detect HLGR, HLSR, and HLAR and help limit the spread of resistance.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
   References Top

1.
MacVane SH, Tuttle LO, Nicolau DP. Demography and burden of care associated with patients readmitted for urinary tract infection. J Microbiol Immunol Infect 2015;48:517-24.  Back to cited text no. 1
    
2.
Murray BE. The life and times of the Enterococcus. Clin Microbiol Rev 1990;3:46-65.  Back to cited text no. 2
    
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Sood S, Malhotra M, Das BK, Kapil A. Enterococcal infections and antimicrobial resistance. Indian J Med Res 2008;128:111-21.  Back to cited text no. 3
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Nelson I, Higuita A, Huycke M. Enterococcal disease, epidemiology, and implications for treatment. In: Gilmore MS, Clewell DB, Ike Y, Shankar N. editors. Enterococci: From Commensals to Leading Causes of Drug Resistant Infection. Boston: Massachusetts Eye and Ear Infirmary; 2014. p. 1-23.  Back to cited text no. 4
    
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Koneman EW, Allen SD, Janda WM, Schreekenberger PC, Winn WC. The gram positive cocci, part II: Streptococci, enterococci, and the “Strepococcus like” bacteria. In: Winn WC, Allen SD, Allen S, Janda WM, Koneman EW, Schreckenberger PC, et al., editors. Colour atlas and textbook of diagnostic microbiology. 6th ed. Philadelphia, USA: Lippincott, Williams & Willkins Publications; 1997. p. 672-764.  Back to cited text no. 5
    
6.
Facklam RR, Collins MD. Identification of Enterococcus species isolated from human infections by a conventional test scheme. J Clin Microbiol 1989;27:731-4.  Back to cited text no. 6
    
7.
In CLSI. Performance Standards for Antimicrobial Susceptibility Testing. Twenty-Third International Supplement. CLSI document M100-S23. Wayne, PA: Clinical and Laboratory Standards Institute; 2013. p. 90-3.  Back to cited text no. 7
    
8.
Shrihari N, Kumudini TS, Karadesai SG, Metgud SC. Speciation of enterococcal isolates and antibiotic susceptibility test including high level aminoglycosides resistance and minimum inhibitory concentration for vancomycin. Int J Biol Med Res 2011;2:865-9.  Back to cited text no. 8
    
9.
Palanisamy S, Karunakaran S, Narayanan S. Antimicrobial resistance profile and characterisation of Enterococcus from various clinical samples in a tertiary care hospital. Int J Med Res Health Sci 2013;2:328-33.  Back to cited text no. 9
    
10.
Parameswarappa J, Basavaraj VP, Basavaraj CM. Isolation, identification, and antibiogram of enterococci isolated from patients with urinary tract infection. Ann Afr Med 2013;12:176-81.  Back to cited text no. 10
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Telkar A, Baragundi MC, Raghavendra VP, Viswanath G. Urinary tract enterococcal infections and their antimicrobial resistance. Int J Pharm Bio Sci 2012;3:90-6.  Back to cited text no. 11
    
12.
Mendiratta DK, Kaur H, Deotale V, Thamke DC, Narang R, Narang P. Status of high level aminoglycoside resistant Enterococcus faecium and Enterococcus faecalis in a rural hospital of central India. Indian J Med Microbiol 2008;26:369-71.  Back to cited text no. 12
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Fernandes SC, Dhanashree B. Drug resistance and virulence determinants in clinical isolates of Enterococcus species. Indian J Med Res 2013;137:981-5.   Back to cited text no. 13
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Bhat KG, Chitra P, Bhat M. High level aminoglycoside resistance in enterococci isolated from hospitalised patients. Indian J Med Res 1997;105:198-9.  Back to cited text no. 14
    
15.
Gordon S, Swenson J, Hill BC, Pigott NE, Facklam RR, Cooksey RC, et al. Antimicrobial susceptibility patterns of common and unusual species of Enterococci causing infections in the United States. Enterococcal Study Group. J Clin Microbiol 1992;30:2373-8.  Back to cited text no. 15
    
16.
Karmarkar MG, Gershom ES, Mehta PR. Enterococcal infections with special reference to phenotypic characterization and drug resistance. Indian J Med Res 2004;119(Suppl):22-5.  Back to cited text no. 16
    

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Correspondence Address:
Smeeta Huidrom
Department of Microbiology, Jawaharlal Nehru Institute of Medical Sciences, Porompat, Imphal, Manipur
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1755-6783.181657

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  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]



 

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