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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 24  |  Issue : 1  |  Page : 12-15

Antibiotic susceptibility pattern in clinical isolates of Enterococci at a tertiary care hospital


1 Student, Department of Microbiology, Geetanjali Medical College and Hospital, Udaipur, Rajasthan, India
2 Assistant Professor, Department of Microbiology, Geetanjali Medical College and Hospital, Udaipur, Rajasthan, India

Date of Submission24-Jan-2022
Date of Acceptance01-Apr-2022
Date of Web Publication11-Jul-2022

Correspondence Address:
Akshat Vij
Assistant Professor, Department of Microbiology, Geetanjali Medical College and Hospital, Hiran Magri Extension, Manwakhera, NH-8 Bypass, Near Eklingpura Chouraha, Udaipur - 313 001, Rajasthan
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jacm.jacm_1_22

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  Abstract 


INTRODUCTION: Enterococci have emerged as leading causes of healthcare-associated infections globally. Glycopeptide-resistant Enterococci have become a major threat to hospitalised patients, causing outbreaks that increase morbidity, mortality and healthcare-associated costs.
MATERIALS AND METHODS: All samples were inoculated on appropriate culture plates. Confirmation was done by performing Gram's stain, catalase test and bile-esculin hydrolysis. Antibiotic susceptibility testing was performed using modified Kirby–Bauer's disk diffusion method. Vancomycin resistance in Enterococci was detected by performing Epsilometer test on Mueller–Hinton agar, and results were interpreted as per CLSI guidelines (2019).
RESULTS: Totally 75 isolates of Enterococci were isolated. The highest resistance in all isolates was against Ciprofloxacin (88%), Erythromycin (81.30%) and Ampicillin (77.33%). The most effective drugs were Linezolid with only 2.66% resistance and Vancomycin with 10.66% resistance. High-level Gentamicin (HLG) resistance was seen in 58.66% of cases and high-level Streptomycin (HLS) resistance was seen in 53.33% of cases. Totally 35 strains showed a combined resistance for both HLG and HLS, which was 46.66%. A total of seven cases were Vancomycin-resistant Enterococci (VRE), 85.6% of which showed high-level Aminoglycoside resistance (HLAR). Resistance for Linezolid was reported at 28.6%.
CONCLUSIONS: Female patients presenting with urinary tract infection are the most common patients with enterococcal infections, with hospitalised patients being more prone, especially in the middle age groups. Appropriate antibiotics need to be instituted after performing antibiotic susceptibility testing to reduce treatment failure arising from HLAR and VRE strains. All VRE isolates should be tested for minimal inhibitory concentration before declaring them Vancomycin resistant.

Keywords: High-level Aminoglycoside resistance, multidrug-resistant organisms, Vancomycin-resistant Enterococci


How to cite this article:
Thakan K, Vij A. Antibiotic susceptibility pattern in clinical isolates of Enterococci at a tertiary care hospital. J Acad Clin Microbiol 2022;24:12-5

How to cite this URL:
Thakan K, Vij A. Antibiotic susceptibility pattern in clinical isolates of Enterococci at a tertiary care hospital. J Acad Clin Microbiol [serial online] 2022 [cited 2022 Aug 11];24:12-5. Available from: https://www.jacmjournal.org/text.asp?2022/24/1/12/350314




  Introduction Top


Evolution of Enterococci from being an intestinal commensal organism of little clinical significance to becoming one of the most common nosocomial pathogens associated with significant morbidity and mortality has taken place over the past few decades.[1],[2]

It has emerged as a super nosocomial infecting pathogen not only due to their inherent resistance to multiple antimicrobial agents (such as Clindamycin, Cephalosporins and Aminoglycosides) but also because they have the capacity to acquire and disseminate determinants of antibiotic resistance (such as Vancomycin-resistance gene clusters).[3] The acquisition of high-level Aminoglycoside resistance and Vancomycin resistance has limited the therapeutic options available for clinicians.[4]


  Materials and Methods Top


All samples except urine were inoculated on blood agar and MacConkey agar by employing quadrant streak plate technique. Urine was plated by semi-quantitative method using a calibrated loop on CLED agar – Cystine Lactose Electrolyte Deficient agar and MacConkey agar. Culture plates were incubated at a temperature of 37°C for 18–24 h and a maximum of 48 h. On obtaining growth on the plates, Gram's stain was performed followed by catalase test. Gram-positive cocci in pairs and giving a negative catalase test were further subjected to bile-esculin hydrolysis test for confirmation that the isolate belonged to the genus Enterococcus.

Antibiotic susceptibility testing was performed according to a predetermined panel of drugs using modified Kirby–Bauer's disk diffusion method, and results were interpreted as per CLSI guidelines (2019).[5] Vancomycin resistance in Enterococci was detected by performing Epsilometer test on Mueller–Hinton agar. The minimal inhibitory concentration (MIC) (in μg/ml) interpretative criteria used for Vancomycin are as follows: ≤4 sensitive, 8–16 intermediate and ≥32 resistant.[5],[6]


  Results Top


A total of 4544 clinical samples were collected from patients and were submitted to the microbiology laboratory. Of these, 2926 (64.39%) samples showed no growth and 1618 (35.61%) were culture positive. Gram-negative pathogens were isolated from 1421 (87.82%) culture-positive samples, whereas Gram-positive cocci were from 197 (12.18%) samples only.

The total number of Gram-positive cocci isolated was 197, of which 75 (38.1%) belonged to Enterococcus spp., 80 (40.6%) were Staphylococcus aureus and 42 (21.3%) were coagulase-negative Staphylococci. Their overall percentage amongst culture-positive (n = 1618) clinical samples was 4.6%, 4.84% and 2.59%, respectively. The most common type of clinical specimen that grew Enterococcus spp. as the pathogen was urine with 55 (73.4%) isolates. Both blood and pus yielded a similar number of isolates, i.e., 10 (13.3%) each.

Fifty-two (69.3%) patients presenting with enterococcal infections were admitted in our hospital. The male-to-female ratio in patients presenting with enterococcal infections was 1:2. When we analysed the division based on the age of the patients, 42 (56%) patients belonged to the age group of 21–60 years while the remaining were children, adolescents and the elderly.

Antibiotic susceptibility pattern was performed by Kirby–Bauer disk diffusion method, and the results are shown in [Table 1].
Table 1: Antibiotic susceptibility pattern of the isolated Enterococci (n=75)

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A combined resistance to both high-level Gentamycin (HLG) and high-level Streptomycin (HLS) was seen in 35 (46.66%) cases. There were seven Vancomycin-resistant Enterococci (VRE) isolated and the maximum isolates were from blood (4), followed by urine (3), and none were from pus samples. The sensitivity patterns of the VRE isolates are shown in [Table 2].
Table 2: Antibiotic susceptibility pattern of Vancomycin-resistant Enterococci (n=7)

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  Discussion Top


We obtained 75 Enterococcus isolates from urine, blood and pus samples. Similar findings have been reported from other studies.[7],[8] The higher number of Enterococci isolated from urine samples is directly proportional to the number of urine samples received in our laboratory. Another attributing factor could be the possible endogenous transfer of Enterococci from the anal region derived from the gut commensals in stool.

Samples yielded Enterococci from 52 (69.30%) specimens collected from patients already admitted in the hospital. Manimala et al.[9] have also reported 69% isolation of Enterococcus from inpatient department (IPD) and 31% isolation from outpatient department (OPD). It is well known that colonised patients in a healthcare setting are a potential source for the spread of pathogenic organisms into their immediate surroundings, to healthcare workers and to other patients. Moreover, hospitalised patients are usually immunologically weak and are prone to acquiring infections from a hospital environment. This could be one of the reasons why we isolated Enterococci from IPD patients more than in patients attending the OPD. Of the 75 Enterococci isolated, 46 (61.33%) were from patients more than 40 years of age. Similar findings have been reported in the study done by Sharma et al.[10] where they found that the maximum percentage of isolates was from patients between the age group of 41 and 60 years. Our study finding of more Enterococcus spp. isolated from the elderly age group is attributable to acquisition of infections and hospitalised proportionally with increasing age.

We isolated a greater number of Enterococci from female patients in comparison to male patients. Similar findings have been reported from other studies.[11] It is noteworthy that out of the 75 isolates in our study, 55 (73.39%) were isolated from urine, and out of those, 34 (61.81%) were isolated from females. The proximity of the urethra to the perianal area due to shorter urethral length in females leads to a higher rate of urinary tract infection (UTI) in them, especially due to Escherichia coli or due to Enterococcus spp. This again reconfirms the known fact that a greater number of UTIs occur in females due to the endogenous transfer of Enterococci.

This study shows that the overall highest resistance in all isolates was against Ciprofloxacin, Erythromycin, Ampicillin and Penicillin. Similar results were reported by Mathew et al.[12] who reported 88% of strains resistant to Ciprofloxacin and 85% of strains resistant to Erythromycin. Yadav et al.[7] also reported 75% resistant enterococcal strains to Ampicillin and Penicillin each. Nitrofurantoin is one of the important and effective drugs for urinary infections. Isolates of Enterococci in our study showed better results in vitro as compared with other studies conducted in India. This is an encouraging finding as it shows that urinary isolates showing resistance to other drugs will be susceptible to Nitrofurantoin and it can be used as an effective therapeutic option in such cases. A similar finding was seen in the study conducted by Atray et al.[8]

The most effective drugs were Linezolid with only 2.66% resistance and Vancomycin with 10.66% resistance. Identifying isolates resistant to Vancomycin is important due to the emergence and spread of VRE strains in the hospital environment. Resistance to high concentration of Aminoglycosides in Enterococci is due to the production of Aminoglycoside-modifying enzymes and gene mutations of antibiotic targets.[13] High-level Gentamicin resistance (HLGR) was seen in 58.66% of cases and HLS resistance (HLSR) was seen in 53.33% of cases. A combined resistance for both HLG and HLS was observed in 35 (46.66%) strains. These findings underscore the importance in detecting HLGR and HLSR because this would directly help limit the nosocomial dissemination of resistance mechanisms and also help to establish a surveillance programme regarding the use of Aminoglycosides for the management of enterococcal infections.[11]

From a total of 75 enterococcal isolates, 67 isolates were sensitive to Vancomycin and 8 (10.66%) were screened as resistant to Vancomycin by disk diffusion method. Out of these 8 isolates, 7 (9.33%) isolates were confirmed as VRE by E-strip test having MIC >64 μg/ml which was similar to the study by Mathew et al.[12] who reported 12.2% VRE by disk diffusion method and 9.62% VRE confirmed by E-strip test. Mittal et al.[14] reported 9% VRE by disk diffusion method and 5% VRE isolates were confirmed by E-strip test. The results of disk diffusion method for Vancomycin sensitivity can be inaccurate. Hence, for the 'final resort' antibiotics such as Vancomycin, MIC monitoring should be done before reporting as intermediate, sensitive or resistant. Although MIC testing for Vancomycin in Enterococci was performed in this study, phenotypic categorisation into Van A, Van B, Van C, etc., could not be performed due to limited resources and cost constraints.

With an inherent Gentamicin resistance and without surveillance of HLGR and HLSR patterns, the emergence of VRE worsens the problem further. The imprudent use of Vancomycin and non-compliance with infection control measures has finally led to the emergence of VRE strains. Multidrug resistance exhibited by these pathogens leaves fewer therapeutic options for clinicians in treating serious life-threatening VRE infections.

High-level Aminoglycoside resistance (HLAR) was seen in 6 (85.7%) of the VRE isolates in our study. Various articles have reported a huge range of resistance patterns for Linezolid over the past decade. A couple of studies have reported 0% resistance to Linezolid.[7],[9] Yadav et al.[7] in 2015 have reported about 42.6% of strains resistant to Linezolid. In our study, resistance for Linezolid was reported at 28.6%.

Emergence of resistance against Linezolid as well as Vancomycin which have been considered last-resort antibiotics against Gram-positive cocci till now is a matter of concern which emphasises the need to explore newer therapeutic options in such conditions. The reasons for these dissimilarities amongst various studies could be the difference in their infection control practices, surveillance for colonisation, identification and isolation amongst colonised patients and strict adherence to hand hygiene practices.


  Conclusions Top


Enterococci are one of the sturdiest pathogens encountered in human infections inherently resistant to Cephalosporins, low-level Aminoglycosides and Clindamycin. We conclude that female patients presenting with UTI are the most common patients with enterococcal infections, with hospitalised patients being more prone, especially in the middle age groups. Appropriate antibiotics need to be instituted after performing antibiotic susceptibility testing to reduce treatment failure arising from HLAR and VRE strains. All VRE isolates should be tested for MIC before declaring them Vancomycin resistant. Establishing a surveillance programme to detect these resistance patterns in Enterococci can provide an edge in defeating infections caused by these pathogens.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Giridhara Upadhyaya PM, Ravikumar KL, Umapathy BL. Review of virulence factors of Enterococcus: An emerging nosocomial pathogen. Indian J Med Microbiol 2009;27:301-5.  Back to cited text no. 1
    
2.
Schouten MA, Hoogkamp-Korstanje JA, Meis JF, Voss A; European VRE Study Group. Prevalence of vancomycin-resistant enterococci in Europe. Eur J Clin Microbiol Infect Dis 2000;19:816-22.  Back to cited text no. 2
    
3.
Arias CA, Murray BE. The rise of the Enterococcus: Beyond vancomycin resistance. Nat Rev Microbiol 2012;10:266-78.  Back to cited text no. 3
    
4.
Karmarkar MG, Gershom ES, Mehta PR. Enterococcal infections with special reference to phenotypic characterization & drug resistance. Indian J Med Res 2004;119 (Suppl):22-5.  Back to cited text no. 4
    
5.
Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing. M100 Standard. 29th ed. Wayne, PA: Clinical and Laboratory Standards Institute; 2019.  Back to cited text no. 5
    
6.
Schulz JE, Sahm DF. Reliability of the E test for detection of ampicillin, vancomycin, and high-level aminoglycoside resistance in Enterococcus spp. J Clin Microbiol 1993;31:3336-9.  Back to cited text no. 6
    
7.
Yadav G, Thakuria B, Madan M, Agwan V, Pandey A. Linezolid and vancomycin resistant enterococci: A therapeutic problem. J Clin Diagn Res 2017;11:C07-11.  Back to cited text no. 7
    
8.
Atray D, Sharma A, Atray M. Prevalence of enterococci and its antibiotic resistance in various clinical samples at tertiary care hospital in Southern Rajasthan, India. Int J Res Med Sci 2017;4:3413-6.  Back to cited text no. 8
    
9.
Manimala E, Rejitha IM, Revathy C. Detection of vancomycin resistant enterococci in various clinical sample isolates from a tertiary care centre. Int J Curr Microbiol Appl Sci 2019;8:915-21.  Back to cited text no. 9
    
10.
Sharma M, Jain S, Bhagat S, Shree N, Kumar M. Antimicrobial resistance pattern of Enterococcus species with special reference to vancomycin resistance. J Microbiol Res 2016;8:775.  Back to cited text no. 10
    
11.
Adhikari RP, Shrestha S, Barakoti A, Rai JR, Amatya R. Antimicrobial susceptibility pattern of Enterococcus species isolated from various clinical specimens in a tertiary care hospital, Kathmandu, Nepal. Nepal Med Coll J 2018;20:173-7.  Back to cited text no. 11
    
12.
Mathew SK. A profile of vancomycin-resistant enterococcal infections and a comparison of resistance detection methods. Indian J Microbiol Res 2018;5:408-14.  Back to cited text no. 12
    
13.
Miller WR, Munita JM, Arias CA. Mechanisms of antibiotic resistance in enterococci. Expert Rev Anti Infect Ther 2014;12:1221-36.  Back to cited text no. 13
    
14.
Mittal S, Singla P, Deep A, Bala K, Sikka R, Garg M, et al. Vancomycin and high level aminoglycoside resistance in Enterococcus spp. in a tertiary health care centre: A therapeutic concern. J Pathog 2016;2016:8262561.  Back to cited text no. 14
    



 
 
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