Join us   Log in   editor.njbms16@gmail.com  


NJBMS - Volume 11, Issue 2, October - December 2020

Pages: 196-200

Date of Publication: 06-Oct-2020


Print Article   Download XML  Download PDF

PHENOTYPIC CHARACTERISATION OF ENTEROCOCCI FROM URINE SAMPLES

Author: Dr. Reena Rajan1, Dr. A. Venkata Raghavendra Rao2, Dr. A. Kavitha3, Dr. S. Mathavi4

Category: Microbiology

Abstract:

Introduction: Enterococci are one of the leading causes of health care associated infection. The clinical significance of Enterococci is directly linked to its resistance traits which contribute to the risk of infection and colonization. Aims & Objectives: The present study aim to detect the phenotypic traits of Enterococci and its antimicrobial resistance pattern. Materials & Methods: 708 isolates of Enterococci from urine samples were identified into species level by sugar utilization test using brain heart infusion broth containing 1% sugars. The Minimum Inhibitory Concentration (MIC) of the isolates to various antibiotics was determined by Vitek 2 automated system. Results: Enterococcus faecalis (93.64%) was the predominant isolate obtained followed by E. Faecium (4.24%), E. avium (1.70%) and E. Durans (0.42%). 13 (1.84%), out of 708 isolates were identified as VRE. Van a phenotype was identified in two isolates of E. faecalis with Vancomycin MIC of 8 and 16μg/ml. Ten isolates of E. faecalis exhibited glycopeptides MIC in the susceptibility range (1-4μg/ml) and showed the presence of Van a phenotype by vitek. Van a phenotype was observed in a single isolate of E. faecium with Vancomycin MIC (? 32μg/ml) and Teicoplanin MIC (32μg/ml). Isolate with Van a phenotype was found to be resistant to Penicillin, High Level Gentamicin, Fluoroquinolones, and Macrolides. Conclusion: Identification to species level is important for appropriate therapy of Enterococcal infection. Phenotypic detection of VRE helps to monitor the resistance pattern of clinical isolates, thereby limiting the spread of bacterial resistance.

Keywords: Enterococcus species, Van Phenotypes, High Level Gentamicin Resistance, Penicillin Resistance.

DOI URL: https://dx.doi.org/10.31975/NJBMS.2020.11203

Full Text:

INTRODUCTION:
Enterococcus is part of normal gut flora of humans and often colonizes oral cavity, genitourinary tract and skin. Nosocomial urinary tract infections (UTI’s) are the most frequent infection caused by these organisms. The predisposing conditions for urinary colonisation and infection by Enterococci rise with patients who have structural abnormalities, recurrent urinary tract infections, frequent instrumentation and prior therapy with antibiotics.1
The antimicrobial therapy of Enterococci infection is complicated due to its innate mechanism of resistance and the ability to develop resistance to broad spectrum antibiotics. Glycopeptide antibiotics are the first choice alternative drug to penicillin- aminoglycoside combination to treat severe Enterococci infection.2
Vancomycin Resistant Enterococci (VRE) are being reported from various parts of the world. High level resistance to glycopeptides is conferred by the modification in the terminal peptidoglycan precursors from D-Ala-D-Ala to D-Ala-D-Lac. Low level resistance is due to peptidoglycan precursors ending in D-Ala-D-Ser, which decreases its binding ability to glycopeptides.2,3
The epidemiology of Enterococci varies with geographical location and Enterococci species varies in their susceptibility pattern to glycopeptides. Hence phenotypic characterization of Enterococci is mandatory for the management of infection.
AIMS & OBJECTIVES:
To isolate and identify Enterococci from urine samples
To determine phenotypic characteristics of Enterococci and their antimicrobial susceptibility pattern.
MATERIALS & METHODS:
TYPE OF STUDY: Cross sectional study
INCLUSION CRITERIA: Patients of all age group isolates of Enterococci in pure culture from urine samples.
EXCLUSION CRITERIA: Non enterococcal infection clinically insignificant isolates of Enterococci. The present study was done at Department of Microbiology from January 2015 to March 2019. A total of 708 isolates collected from inpatients and outpatients at VIMS hospital and VMKV Medical College, Salem were included in this study. A loopful of the sample was inoculated onto Blood agar and MacConkey agar. The plates were incubated at 370C overnight for 24-48 hours.4,5A colony count of more than 105 CFU / ml was considered as significant bacteriuria. Antimicrobial susceptibility testing of Enterococci was determined by Vitek 2 automated system according to manufacturer’s instructions. Detection of Minimum inhibitory concentrations was performed for Penicillin, Erythromycin, Vancomycin, Teicoplanin, Ciprofloxacin, Tigecycline, Tetracycline, Nitrofurantoin, Linezolid and Daptomycin. 6Isolates were identified as genus Enterococcus based on gram staining, colony morphology, catalase test, growth in 6.5% Nacl and bile esculin agar, heat tolerance test etc.
IDENTIFICATION OF GENUS ENTEROCOCCUS:
GRAM STAIN: From the solid media, small portion of colonies were picked up with a loop and mixed on a slide with loopful of sterile normal saline to form a light suspension. Stain was applied to heat fixed smear. Presence of gram positive oval cocci in pairs and short chains indicated the presence of Enterococci.
Dr. Reena Rajan. et al., Phenotypic characterisation of Enterococci from urine samples
National Journal of Basis Medical Science / Volume 11 / Issue 2 / 2020 Oct-Dec 197
CATALASE TEST: A part of colony was emulsified in saline on a clean slide and a drop of 3 % hydrogen peroxide was added to the suspension. A positive test is indicated by the appearance of effervescence. No effervescence confirmed the absence of enzyme
TOLERANCE WITH 6.5% NACL: The colonies were inoculated in nutrient broth with 6.5% NaCl, and incubated at 370C for 3 days. The tubes were checked for any increase in turbidity and further confirmed by sub-culturing onto blood agar plate. Turbidity confirmed the presence of organism, indicated the ability of Enterococci to grow in this concentration.4,5
HEAT TOLERANCE TEST:
Enterococci were inoculated in nutrient broth and incubated in water bath set at 100C and 450C for half an hour, and sub-cultured on blood agar plate. Enterococci survived in both the temperature showed positive reaction.4,7
BILE ESCULIN TEST: Colonies from overnight culture was inoculated in media containing 40% bile. Blackening of the medium confirmed the positive reaction.7
IDENTIFICATION OF ENTEROCOCCUS SPECIES:
MOTILITY TEST: Stab culture of the colony was done in a semi-solid media and motility was indicated by the spread of the organism into the media. The strains which did not diffuse into semisolid media were considered non-motile. Tubes were incubated for upto 7 days for confirming motility.8
ARGININE TEST: A well isolated colony of Enterococcus from the culture was inoculated in Moeller’s arginine decarboxylase media overlaid with mineral oil and incubated at 370C for 48 hours. The change in colour of the tube to purple indicated positive reaction. Yellow colour indicated only acid production and no deamination.8
POTASSIUM TELLURITE REDUCTION TEST: Colonies were inoculated onto media containing 0.04% potassium tellurite and incubated for 48 hours. E. faecalis formed black colonies with the reduction of tellurite to tellurium.5
PIGMENT PRODUCTION: Ability to produce pigment was tested by sub culturing colonies in nutrient agar and any colour produced was further confirmed by touching the colony with a sterile swab.8
SUGAR FERMENTATION TEST: Species identification was further done by sugar utilization by inoculating the organism in brain heart infusion broth containing 1% sugars with Bromothymol blue indicator. The sugars used were Mannitol, Sorbitol, Sorbose, Arabinose, Raffinose, Lactose and Sucrose. Any change in colour from green to yellow indicated positive for sugar fermentation. Absence of colour change was taken as negative.8
RESULTS:
Among the Enterococci isolates from urine 663 were Enterococcus faecalis, 30 were E. faecium, 12 were E. avium and 3 were E. durans. (Fig: 1)
Fig 1: Distribution of Enterococcus species from urine sample
93.64
93.64
4.24
4.24
1.7
1.7
0.42
0.42
Percentage distribution of
Percentage distribution of Enterococcus speciesEnterococcus species
E.faecalis
E.faecalis
E.faecium
E.faecium
E.avium
E.avium
E.durans
E.durans
Dr. Reena Rajan. et al., Phenotypic characterisation of Enterococci from urine samples
National Journal of Basis Medical Science / Volume 11 / Issue 2 / 2020 Oct-Dec 198
69 (10.41%) isolates of E. faecalis and 6 (20.00%) isolates of E. faecium were resistant to Penicillin with MIC of 16μg/ml.399 (60.18%) isolates of E. faecalis and 21(70.00%) isolates of E. faecium, were resistant to Ciprofloxacin with MIC ? 8μg/ml. 6 (0.90%) isolates of E. faecalis from urine were resistant to Linezolid with MIC ? 8μg/ml. [Table: 1]
Among the isolates resistant to Nitrofurantoin, 111 (16.74%) were E. faecalis with MIC of >128μg/ml and 12 (1.81%) isolates of E. faecalis showed MIC of 256μg/ml. 6 (20.00%) isolates of E. faecium were resistant with MIC of 128μg/ml.
Table 1: Detection of antimicrobial resistance among Enterococci isolates by MIC method.
Antibiotics Susceptible Resistant
Penicillin
577(81.50%)
131(18.50%)
High Level Gentamicin
312(44.07%)
396(55.93%)
Ciprofloxacin
252(35.59%)
456(64.41%)
Erythromycin
126(17.80%)
582(82.20%)
Linezolid
702(99.15%)
6(0.85%)
Nitrofurantoin
480(67.80%)
228(32.20%)
Vancomycin
705(99.58%)
3(0.42%)
Teicoplanin
707(99.86%)
1(0.14%)
537 (81.00%) isolates of E. faecalis showed MIC of 0.5μg/ml for Teicoplanin. The Teicoplanin MIC for 29/30 (96.67%) isolates of E. faecium were 0.5μg/ml and one isolate showed MIC of 32μg/ml.
13 (1.84%), out of 708 isolates were identified as VRE. Van A phenotype was identified in two isolates of E. faecalis with Vancomycin MIC of 8 and 16μg/ml. Ten isolates of E. faecalis exhibited glycopeptide MIC in the susceptibility range (1-4μg/ml) and showed the presence of Van A phenotype by vitek. [Fig: 2] Van A phenotype was observed in a single isolate of E. faecium with Vancomycin MIC (? 32μg/ml) and Teicoplanin MIC (32μg/ml). [Fig: 3] (1-32μg/ml) by Vitek
Fig 2: Detection of E. faecalis phenotypes
Fig 3: Detection of E. faecium pheno types by Vitek
DISCUSSION:
The identification of Enterococcito species level is essential as each species varies in their resistance to antibiotics and species level identification may be useful for epidemiologic surveillance within hospitals. In the present study the most common species isolated was E. faecalis (93.64%) followed by E. faecium (4.24%) and E. avium (1.70%). A study finding by PJ Desai et al., have showed 49.50% of E. faecalis and 35.64% E. faecium isolates from urinary tract infection.9 A study conducted by N Taneja et al. , have reported Enterococcus faecalis (55%) as predominant isolate from urine sample.10
The penicillin MIC for 10.41% E. faecalis and 20.00% isolates of E. faecium were 16μg/ml. In a similar study, M R Maradia
2
2
1
1
8
8
16
16
4
4
4
4
4
4
4
4
0
0
2
2
4
4
6
6
8
8
10
10
12
12
14
14
16
16
18
18
7
7
3
3
1
1
1
1
Minimum Inhibitory
Minimum Inhibitory ConcentrationConcentration
No of Isolates with MIC range 1
No of Isolates with MIC range 1--32µg/ml 32μg/ml for vancomycin and teicoplanin for vancomycin and teicoplanin
Vancomycin MIC
Vancomycin MIC
Teicoplanin MIC
Teicoplanin MIC
32
32
32
32
0
0
10
10
20
20
30
30
40
40
1
1
Minimum Inhibitory
ConcentrationConcentration
No of Isolates
No of Isolates
Vancomycin MIC
Vancomycin MIC
Teicoplanin MIC
Teicoplanin MIC
Dr. Reena Rajan. et al., Phenotypic characterisation of Enterococci from urine samples
National Journal of Basis Medical Science / Volume 11 / Issue 2 / 2020 Oct-Dec 199
et al., have reported 97.44% isolates from urine resistant to Penicillin.11 In a North Indian study by N Gangurde et al, E. faecium isolates exhibited significantly high resistance to penicillin as compared to E. faecalis.12 In the present study no statistically significant difference in resistance to Penicillin was found between E. faecalis and E. faecium isolates (p value >0.05).
In the present study, 14.93% E. faecalis were resistant to Nitrofurantoin with MIC of 128μg/ml. In a study from South India by K Suresh et al., 100% isolates showed susceptibility to Nitrofurantoin.13 A similar study by D A tray et al., have reported 80% urinary isolates susceptible to Nitrofurantoin.14
The present study reports two isolates of Enterococcus faecalis showing intermediate resistance to Vancomycin. Van A phenotype was identified in two isolates of E. faecalis with Vancomycin MIC of 8 and 16μg/ml. Presence of Van A phenotype among E. faecalis have been reported from various Indian studies.15,16,17,18. Enterococcus faecium isolate with Van a phenotype was found to be resistant to Penicillin, High Level Gentamicin, Fluoroquinolones, and Macrolides.
CONCLUSION: There has been a rapid increase in the incidence of VRE infection due to extended use of Vancomycin and extended spectrum Cephalosporins in health care settings. The ability of these organisms to transfer Vancomycin-resistance genes to other gram positive organisms is a cause of clinical concern. Phenotypic characterisation of Enterococci is essential to detect low-level glycopeptide resistance and to distinguish between the different Van phenotypes.
SOURCES OF FUNDING: Nil
CONFLICTS OF INTEREST: Nil
ACKNOWLEDGEMENT: I thank Dr. V. Anandi, Former Professor, Department of Microbiology, Vinayaka Mission’s Medical College Karaikal, Tamil Nadu for guiding me in my research work. We are extremely grateful to VIMS hospital staff for the support extended towards successful completion of research work.

References:

1. S Sood, M Malhotra, B K Das and A Kapil, “Enterococcal infections and antimicrobial resistance,” Indian J Med Res,2008; 128 (2):111-121.
2. W R Miller, J M Munita and C AArias, “Mechanism of antibiotic resistance in Enterococci,” Exp Rev Anti Infect Ther, 2014;12 (10): 1221-1236
3. S Sujatha and I Praharaj, “Glycopeptide resistance in gram positive cocci: A Review,” Interdisciplinary perspectives on Infectious diseases;2012: 1-10.
4. R S Miles and S G B Amyes, “Laboratory control of antimicrobial therapy,” Chapter 8. In: ColleeJG, Fraser AG, Marmion BP, Simmons A, editors, Mackie and McCartney Practical Medical Microbiology, 14 th ed. London:Churchill Livingstone; 1996: 151-78
5. R RFacklam and R D Carey, The Streptococci and Aerococci, In E. H. Lennette, A. Balows, W. J. HauslerJr., and H.J.Shadomy(ed.), Manual of Clinical Microbiology, 4th ed. American Society for Microbiology, Washington D. C. 1985 : 154-175.
6. Clinical and Laboratory Standard Institute (CLSI). Performance standards for antimicrobial susceptibility testing 26th ed. Wayne USA. CLSI, 2016
7. E W Koneman, S D Allen, W M Janda, P C Schreckenberger and W C Winn, “Isolation and Identification Streptococci
Dr. Reena Rajan. et al., Phenotypic characterisation of Enterococci from urine samples
National Journal of Basis Medical Science / Volume 11 / Issue 2 / 2020 Oct-Dec 200
like organisms” Koneman’s Colour Atlas and Text Book of Diagnostic Microbiology,”6th ed; Baltimore Lippincott William and Wilkins. 2006:726-33.
8. R RFacklam and M D Collins, “Identification of enterococcal species isolated from human infections by a conventional test scheme,” J ClinMicrobiol, 1989: 27 (4): 731-4.
9. P J Desai, D Pandit, M Mathur, and A Gogate,“Prevalence, Identification and distribution of various species of Enterococci isolated from clinical specimens with special reference to UTI in catheterised patients, ” Indian J Med Microbiol, 2001: 19 (3): 132-137.
10. N Taneja, P Rani, R Emmanuel and M Sharma, “Significance of Vancomycin resistant Enterococci from urinary specimens at a tertiary care centre in northern India, ”Indian JMed Res, 2004 : 119: 72-4.
11. M R Maradia, K Mehta, K Prajapati, M Vadsmiya, P Shah, M Vegad, “Prevalence of multidrug – resistant Enterococcus species isolated from urine samples in a tertiary care hospital, Western India, ” Int J Med Sci Public Health, 2017; 6 (4):1-5. 12. N Gangurde, M Mane and S Phatale, “Prevalence of Multidrug Resistant Enterococci in a Tertiary Care Hospital in India: A Growing Threat,” Open J Microbiol,2014; 4 (1):11-15.
13. K Suresh, B Saipriya and G Viswanath, “Isolation, Speciation and Determination of High level Aminoglycoside resistance of Enterococci among hospitalised patients in Davangere, ” National J Lab Med, 2013,2 (1): 12-15 14. D Atray, A Sharma and M Atray, “Prevalence of Enterococci and its antibiotic resistance in various clinical samples at tertiary care hospital in Southern Rajasthan India,” Int J Res Med Sci, 2016; 4 (8):3413-3416
15. P Mathur, A Kapil, R Chandra, P Sharma and B Das, “Antimicrobial resistance in Enterococcus faecalis at a tertiary care centre of Northern India,” Indian J Med Res,2003; 118: 25-28.
16. U Ghoshal, A Garg, D P Tiwari, A Ayyagiri, “Emerging Vancomycin resistance in Enterococci in india,” Indian J Pathol Microbiol, 2006;49 (4):620-622.
17. S Mulla, K G Patel, T Panwala and S Rewadiwala, “Prevalence of Enterococci with higher resistance level in a tertiary care hospital-A matter of concern,” National J Med Res, 2012;2 (1): 25-27.
18. G B Modi, S T Soni, J Patel, H M Goswami and M MVegad, “Prevalence of Vancomycin Resistant Enterococci in Tertiary Care Hospital, Western India,” Int J Microbiol Res, 2012;4 (2): 182-185.