INTRODUCTION
Enterococcus species are normal inhabitants of intestinal tracts of healthy humans and animals but have the potential to cause opportunistic infections, especially in immunocompromised patients.[1] They are catalase negative, Gram-positive cocci, classified under group D streptococcus, typically oval shaped and occur in singles, pairs, and short chains.[2] The genus Enterococcus includes more than 50 species; Enterococcusfaecalis and Enterococcusfaecium are being the two most recognized species associated with hospital-associated infections.[3,4] They are sturdy organisms which can thrive in a range of stressful and hostile environments including ability to survive desiccation, grow in the presence of 40% bile salts, 6.5% NaCl, high pH, and wide temperature range of 10°C–45°C.[3–5] These attributes make Enterococci prolific colonizers with an immense genome plasticity and a tendency for persistence in hospital environment. Thus, allowing transmission and dissemination of resistant strains among hospitalized patients.[5,6] In the past two decades, Enterococci have emerged as one of the major nosocomial pathogens and are associated with wide range of hospital-acquired infections such as catheter-associated urinary tract infection (CAUTI), surgical and burn wound infection, bacteremia, neonatal sepsis, endocarditis, and less commonly, central nervous system infections.[2,7,8]
Treatment of Enterococcus infections is challenging because of their intrinsic as well as acquired resistance to many common antimicrobial agents.[1] They carry several resistance genes that encode for intrinsic resistance to number of antimicrobial agents such as aminoglycosides and beta-lactams.[3,9] In addition, they acquire resistance against several antibiotics such as macrolides, fluoroquinolones, tetracycline, cephalosporin, linezolid, and vancomycin, either by DNA mutation or acquisition of new resistant-genes, including those which encode for beta-lactamase production through gene transfer mechanisms.[1,10–12] Prolonged length of hospital stays and indiscriminate use of antibiotics are the two most recognized contributing factors for emergence and dissemination of drug-resistant Enterococci. Prolonged use of broad-spectrum antibiotics dramatically results in depletion of normal gut flora and assist Enterococci to outcompete the normal Gram-negative gut microbiota and overgrow in addition to selective antibiotic pressure and acquisition of resistance.[1,13] Recent studies have reported increase in emergence of vancomycin-resistant Enterococci (VRE) worldwide, posing further challenge to treatment.[14] The prevalence of Enterococcus spp. infection, rate, and pattern of antimicrobial resistance among Enterococcus spp. has a wide geographic variation.[15] Therefore, accurate knowledge regarding local data of prevalence of Enterococci spp. and their antimicrobial resistance pattern among clinician would assist them greatly in proper management of infections. As far as our knowledge, there were no studies related to frequency of Enterococci infection and their resistance pattern in northern region of Oman. Hence, this study was aimed to investigate clinical characteristics and determination of antimicrobial susceptibility pattern of E. faecalis and E. faecium with an emphasis on vancomycin resistance during the study period from January 2017 to December 2021.
METHODS
This retrospective study was conducted at Sohar hospital, Oman. The study was approved by the Institutional Ethic and Review committee as well as from the Ministry of Health, Oman (Approval number: MH/DGHS/NBG/RERAC22/2022). All Enterococci identified as E. faecium and E. faecalis during the period from January 2017 to December 2021 were included in the study. The data of the patients diagnosed with E. faecalis and E. faecium infection were extracted from the Sohar Hospital’s electronic health records. The data included the demographics, relevant clinical details, and microbiological data including antibiotic susceptibility pattern of E. faecium and E. faecalis. All the information were entered to a predesigned pro forma. Confidentiality of the patient details was strictly maintained.
Inclusion criteria
E. faecalis and E. faecium recovered from clinical samples such as pus, blood, urine, and sterile body fluids were included in the study.
Exclusion criteria
Enterococcusspecies isolated from sputum, skin, stool, and throat swabs in the absence of clinical symptoms was considered asymptomatic colonization and excluded from the study.
Sample collection and identification of Enterococcus species
Clinical samples received at the Sohar Hospital’s microbiology laboratory for culture and sensitivity were inoculated onto MacConkey agar and blood agar plates and incubated at 37°C for 18–24 h according to the CLSI guidelines.[16] Pure isolates were preliminarily characterized by colony morphology and Gram-stain. An isolate is identified as genus Enterococci by catalase, Bile–Esculin agar, culture on nutrient broth at 10°C and 45°C, and growth in the presence of 6.5% NaCl. Speciation into E. faecalis and E. faecium was done by performing sugar fermentation tests (L-arabinose, mannitol, sorbitol glycerol D-lyxose, galactose, and hippurate), arginine dihydrolase, and pyruvate utilization tests as per the standard CLSI guidelines.[16]
Antibiotic susceptibility testing
Antibiotic susceptibility testing was carried out for the isolates by Kirby–Bauer disk diffusion technique on Mueller–Hinton agar as per the CLSI guidelines using antibiotic disks procured from Oxoid Ltd., United Kingdom.[16] Antimicrobial disks used for determining antibiotic susceptibility were erythromycin (15 μg), ampicillin (10 μg), amoxicillin-clavulanic (30 μg), Cefepime (30 μg), vancomycin (30 μg), clindamycin (2μg), cefotaxime (30 μg), cefuroxime (30 μg), amoxicillin-clavulanic acid (30 μg), ciprofloxacin (5 μg), linezolid (30 μg), vancomycin (30 μg), nitrofurantoin (300 μg), and linezolid (10 μg). Mueller–Hinton agar plates were inoculated with 0.5 McFarland standard suspension of the Enterococci strains, then antimicrobial disks were placed into plates and then plates were incubated at 37°C for 24 hour. The zone of inhibition was interpreted as susceptible (S), intermediate (I), or resistant (R) as per the standard guidelines of CLSI.[16] Vancomycin resistance was screened by disk diffusion method and the resistant isolates were further confirmed by agar dilution method.[16] Quality control was performed using E. faecalis ATCC 29212 strain.
Statistical analysis
The data were coded and entered into the Excel file. Data cleaning was performed and all data with incomplete values were filtered out. Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS), IBM Chicago, IL, USA, version 26. The quantitative variables were expressed as mean ± standard deviation, while qualitative variables presented as frequencies. Chi-square test, Student’s t-test, and other relevant tests were carried out for further data analysis and associations.
RESULTS
A total of 132 nonduplicate Enterococcusspecies identified as E. faecalis and E. faecium during 2017–2021 were included in the study. Table 1 demonstrates demographic characteristics of patients infected with Enterococcusspecies. Enterococcus was isolated more commonly from females (54.5%) compared to males (45.5%). The frequency of isolation was highest (47.7%) among elderly people of age >60 years. E. faecalis (68.9%) was the most frequently isolated species, followed by E. faecium (31.1%).
;)
Table 1: Demographic characteristics of study population
Figure 1 summarizes the frequency of isolation of E. faecalis and E. faecium from different clinical samples. The highest frequency of isolation was from urine (49.6%), followed by blood (29.1%), pus/wound swab (11.8%), soft tissue (4.7%), 1.6% each from eye and ear swabs, and 0.8% each from catheter tip and endotracheal tube secretions.
Figure 1: Frequency of isolation of Enterococcus faecalis and Enterococcus faecium from different clinical samples
Figure 2 summarizes antibiotic susceptibility pattern of E. faecalis and E. faecium. All tested E. faecalisandE. faecium were susceptible to linezolid, while all strains were resistant to clindamycin. None of the E. faecalis isolates exhibited resistance to vancomycin, while 5 (13.5%) of the E. faecium isolates were resistant to vancomycin. E. faecalis demonstrated higher susceptibility to ampicillin (92%), amoxicillin–clavulanic acid (93.2%), nitrofurantoin (95.5%), penicillin G (57.1%), and ciprofloxacin (50%). By contrast, E. faecium showed low susceptibility to ampicillin (5%), amoxicillin–clavulanic acid (8.7%), ciprofloxacin (2.8%), and nitrofurantoin (16.6%). All tested E. faecium strains showed resistance to erythromycin, cefotaxime, cefuroxime, and ceftriaxone. E. faecalis also showed low susceptibiltity to these antibiotics ranging 18%-33%.
Figure 2: Antibiotic susceptibility pattern (%) of the Enterococcus faecalis and Enterococcus faecium, AMPI: Ampicillin, AUGM: Amoxicillin-clavulanic acid, CFXM: Cefuroxime, CFTX: Cefotaxime, ERYT: Erythromycin, CIPR: Ciprofloxacin, CLIN: Clindamycin, CRONM: Ceftriaxone, LINZ: Linezolid, NITR: Nitrofurantoin, PENGM: Penicillin G, VANC: Vancomycin
DISCUSSION
The present study was conducted to determine clinical infection and antibiotic profile with an emphasis on vancomycin resistance among E. faecalis and E. faecium isolated at a tertiary care hospital in Northern region of Oman. Enterococci are currently considered one of the major nosocomial pathogens and are increasingly demonstrating more resistant to antimicrobial agents.[17] Almost all hospital-associated infections are presently caused by either E. faecalis or E. faecium. E. faecalis is the most common pathogenic species. However, E. faecium is also gaining importance as it is the second most frequently isolated species and demonstrates more resistance to antimicrobial agents.[2,9]
Commonly, Enterococcus are involved in a variety of nosocomial infections such as CAUTI, bacteremia, endocarditis, neonatal sepsis, surgical and burn wound infection, pneumonia, and more rarely meningitis. In the present study, we observed that maximum number of Enterococci were isolated from urine followed by blood, wound swab/pus, and tissue fragments, which is consistent with the findings of previous studies.[2,18]E. faecalis and E. faecium were the most frequently isolated species in our study, comparable with the previous studies which have reported E. faecalis as the predominant species and E. faecium as the second most common species isolated in clinical samples. Furthermore, Enterococci were more frequently (53.1%) isolated from females and in elderly people aged >60 years (44.6%). These findings were in agreement with the reports of previous studies.[2,19,20]
Antimicrobial resistance among Enterococci for the commonly used antibiotics is currently spreading globally. Enterococcispecies have inherent tenacity to develop resistance to antibiotics and environmental stressors that facilitate them to thrive in hospital environment. Furthermore, continual overuse of antibiotics has driven the evolution of multidrug-resistant (MDR) strains.[4] In the present study, E. faecalis and E. faecium had an overall MDR rate of 7.7% and 58.5%, respectively. This finding is in line with a study by Abera etal.[17] All Enterococcus isolates, in the present study, exhibited susceptibility to linezolid. This result was in agreement with studies from India and Bangladesh which reported zero resistance among Enterococci to linezolid.[20–22] Overall, E. faecium has shown high-level resistance (>90%) to beta-lactams, erythromycin, and ciprofloxacin in the present study. By contrast, E. faecalis exhibited low resistance (<10%) to ampicillin, amoxicillin–clavulanic acid, and nitrofurantoin. This finding is congruent to the previous literature.[18,20–22]
Unanticipated rapid spread and steady increase in VRE worldwide is challenging as they are difficult to treat due to limited treatment option. Moreover, wide geographic variation in vancomycin resistance rate among Enterococci was observed. Most VRE outbreaks are due to acquisition of vancomycin resistance genes such as vanA or vanB among hospital strains.[23] In our study, low vancomycin resistance was observed among E. faecium (14.5%) and none of the E. faecalis exhibited resistance to vancomycin. A recent surveillance report indicates diverseness in the prevalence of vancomycin resistance worldwide. A multicentric study reported high rate of vancomycin resistance in E. faecium (47%) compared to E. faecalis (3%). Furthermore, region-wise distribution showed wide variation with the highest vancomycin resistance among E. faecium (76%) and E. faecalis (5.6%) in the United States, followed by Latin America (48% and 3%), Europe (31.5%, 1.5%), and Asia Pacific (14%, 0.01%).[24] Recent surveillance report in Europe reported an increasing proportion of vancomycin resistance among invasive isolates of E. faecium.[25] The possible explanation for the geographic differences in vancomycin resistance might be due to the gradual change in the MDR strains, selective antibiotic pressure, difference in antibiotic prescription, standards of infection prevention measures, and socioeconomic status. Furthermore, vancomycin resistance would result in coresistance or cross-resistance to range of antibiotics used as alternative agents for vancomycin-resistant E. faecium infections. Ayobami etal. reported that vancomycin-resistant E. faecium have exhibited higher rate of coresistance to ampicillin, amoxicillin, and gentamicin as compared to vancomycin-sensitive E. faecalis. The study also reported notably low-level cross resistance among VRE strains to linezolid, an alternative drug used for treating VRE infection.[26] In line with this finding, the present study demonstrated higher resistance among E. faecium to beta-lactams and most other commonly used antibiotics except linezolid. From the above findings, it is implicated that antibiotic prescription policy and standard infection control measures would greatly help in preventing emergence and spread of drug-resistant strains including vancomycin resistance and associated coresistance and cross-resistance among Enterococci strains.
Limitation of study
There are few limitations in this study that could be addressed in future research. First, the sample size was small and from a single center. Second, the study did not differentiate between hospital-associated infection and community-acquired infection. Third, risk factors including length of hospital stay and outcome of infection were not assessed. Finally, vancomycin resistance genes were not determined.
CONCLUSIONS
Our study reveals the high degree of resistance to macrolide and fluoroquinolone among the nosocomial Enterococcal isolates, thereby limiting the use of these drugs for therapeutic purposes. E. faecalis and E. faecium are the two most common species inherently resistant to cephalosporins and clindamycin, but highly susceptible to linezolid and vancomycin. However, therapy with linezolid or vancomycin should be restricted for treating patients infected with MDR strains only. Judicious use of vancomycin and linezolid in serious infections, appropriate infection control measures, and antibiotic prescription after performing antibiotic susceptibility testing would probably recede the possible emergence of MDR Enterococci outbreaks.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
Acknowledgments
The authors express their gratitude to Mr. Jaya Prasad, an Information and Technology department of Sohar Hospital and all the microbiology laboratory staff of Sohar Hospital for their assistance in data collection and completing this study.
REFERENCES
1. Ramos S, Silva V, Dapkevicius ML, Igrejas G, Poeta P. Enterococci, from harmless bacteria to a pathogen. Microorganisms 2020;8:1118.
2. Sumangala B, Sharlee R, Sahana Shetty NS Identification of
Enterococcus. faecalis and
E. faecium among enterococci isolated from clinical samples in a teaching hospital Mandya Institute of Medical Sciences, Mandya. Indian J Microbiol Res 2020;7:284–7.
3. Fiore E, Van Tyne D, Gilmore MS. Pathogenicity of Enterococci. Microbiol Spectr 2019;7 4:10. doi:10.1128/microbiolspec. GPP3-0053-2018.
4. Zhou X, Willems RJ, Friedrich AW, Rossen JW, Bathoorn E.
Enterococcus faecium:From microbiological insights to practical recommendations for infection control and diagnostics. Antimicrob Resist Infect Control 2020;9:130.
5. García-Solache M, Rice LB. The Enterococcus:A Model of Adaptability to Its Environment. Clin Microbiol Rev 2019;32:e00058–18. doi:10.1128/CMR.00058-18.
6. O'Driscoll T, Crank CW. Vancomycin-resistant enterococcal infections:Epidemiology, clinical manifestations, and optimal management. Infect Drug Resist 2015;8:217–30.
7. Esmail MA, Abdulghany HM, Khairy RM. Prevalence of Multidrug-Resistant Enterococcus faecalis in Hospital-Acquired Surgical Wound Infections and Bacteremia:Concomitant Analysis of Antimicrobial Resistance Genes. Infect Dis (Auckl) 2019;12:1178633719882929. doi:10.1177/1178633719882929.
8. Dubin K, Pamer EG. Enterococci and Their Interactions with the Intestinal Microbiome. Microbiol Spectr 2014;5:10.1128/microbiolspec. BAD-0014-2016. doi: 10.1128/microbiolspec.BAD-0014-2016.
9. Hollenbeck BL, Rice LB. Intrinsic and acquired resistance mechanisms in enterococcus. Virulence 2012;3:421–33. doi:10.4161/viru.21282.
10. Johnston LM, Jaykus LA. Antimicrobial resistance of
Enterococcus species isolated from produce. Appl Environ Microbiol 2004;70:3133–7.
11. Ahmed MO, Baptiste KE. Vancomycin-resistant enterococci:A review of antimicrobial resistance mechanisms and perspectives of human and animal health. Microb Drug Resist 2018;24:590–606.
12. Egan SA, Corcoran S, McDermott H, Fitzpatrick M, Hoyne A, McCormack O, et al. Hospital outbreak of linezolid-resistant and vancomycin-resistant ST80
Enterococcus faecium harbouring an optrA-encoding conjugative plasmid investigated by whole-genome sequencing. J Hosp Infect 2020;105:726–35.
13. Aslam B, Wang W, Arshad MI, Khurshid M, Muzammil S, Rasool MH, et al. Antibiotic resistance:A rundown of a global crisis. Infect Drug Resist 2018;11:1645–58.
14. Melese A, Genet C, Andualem T. Prevalence of vancomycin resistant enterococci (VRE) in Ethiopia:A systematic review and meta-analysis. BMC Infect Dis 2020;20:124.
15. Pfaller MA, Cormican M, Flamm RK, Mendes RE, Jones RN. Temporal and geographic variation in antimicrobial susceptibility and resistance patterns of enterococci:Results from the SENTRY antimicrobial surveillance program, 1997-2016. Open Forum Infect Dis 2019;6:S54–62.
16. Performance Clinical and Laboratory Standards Institute. Standards for Antimicrobial Susceptibility Testing. CLSI Supplement M100. After M100, kindly add Wayne, PA: Clinical and Laboratory Standards Institute; 2021.
17. Abera A, Tilahun M, Tekele SG, Belete MA. Prevalence, Antimicrobial Susceptibility Patterns, and Risk Factors Associated with Enterococci among Pediatric Patients at Dessie Referral Hospital, Northeastern Ethiopia. Biomed Res Int 2021;2021:5549847. doi:10.1155/2021/5549847.
18. Ferede ZT, Tullu KD, Derese SG, Yeshanew AG. Prevalence and antimicrobial susceptibility pattern of Enterococcus species isolated from different clinical samples at Black Lion Specialized Teaching Hospital, Addis Ababa, Ethiopia. BMC Res Notes 2018;11:793.
19. Tripathi A, Shukla SK, Singh A, Prasad KN. Prevalence, outcome and risk factor associated with vancomycin-resistant
Enterococcus faecalis and
Enterococcus faecium at a tertiary care hospital in Northern India. Indian J Med Microbiol 2016;34:38–45.
20. 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.
21. Chakraborty A, Pal NK, Sarkar S, Gupta MS. Antibiotic resistance pattern of enterococci isolates from nosocomial infections in a tertiary care hospital in Eastern India. J Nat Sci Biol Med 2015;6:394–7.
22. Islam TA, Shamsuzzaman SM. Isolation and species identification of enterococci from clinical specimen with their antimicrobial susceptibility pattern in a tertiary care hospital, Bangladesh. J Coast Life Med 2015;3:787–90.
23. Freitas AR, Sousa C, Novais C, Silva L, Ramos H, Coque TM, et al. Rapid detection of high-risk
Enterococcus faecium clones by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Diagn Microbiol Infect Dis 2017;87:299–307.
24. Cattoir V, Leclercq R. Twenty-five years of shared life with vancomycin-resistant enterococci:Is it time to divorce?. J Antimicrob Chemother 2013;68:731–42.
25. Surveillance of Antimicrobial Resistance in Europe; 2016. Available from:
https://ecdc.europa.eu/sites/portal/files/documents/AMR-surveillance-Europe-2016.pdf.2017. [Last accessed on 2023 Mar 06].
26. Ayobami O, Willrich N, Reuss A, Eckmanns T, Markwart R. The ongoing challenge of vancomycin-resistant
Enterococcus faecium and
Enterococcus faecalis in Europe:An epidemiological analysis of bloodstream infections. Emerg Microbes Infect 2020;9:1180–93.
