Study on the Prevalance, Identification and Antibiogram of Enterococci Isolated from the Heterogenous Clinical Samples in a Tertiary Care Hospital

The rise in Pantone-Valentine leucocidin (PVL) and gAMMA-haemolysin (Hlg) producing Enterococcus spp., among the people is a serious concern on evolution of antibiotic resistance. Recent reports suggested that the synergistic association of PVL toxin and necrotic lesions that lead to infections in skin and mucosa. To study the prevalence of Enterococcus isolated from the various clinical samples from both inpatient and out patient department at a Tertiary care hospital. The resistance to Pantone-Valentine leucocidin (PVL) and gAMMA-haemolysin (Hlg) is further tested by using E-test strips for gentamicin test showed 26.8% of isolated populations had resistance to cell wall active agents. In our study all the intermediately sensitive to vancomycin isolates were found to be vancomycin sensitive by both the Epsilometer test and automated Vitek 2 system. The minimum inhibitory concentration ranging from 0.5 4 ug/ml. Hence my study confirmed good activity of the vancomycin, linezolid and Teicoplanin against isolated Enterococci spp. Original Research Article Gulzar and Saikumar; JPRI, 33(25B): 26-34, 2021; Article no.JPRI.67497 27


INTRODUCTION
The term "enterococcus" has originated from the Greek word enteron meaning "the gut or intestine" and kukkos that means "kernel or a berry". These are Gram-positive cocci arranged in pairs and short chains, facultative anaerobic organisms that are a part of normal flora of the oral cavity, gastrointestinal tract and genitourinary tract in humans and animals [1,2]. They were previously classified as group D streptococci but were recognized as a separate entity in 1984 by Schleifer and Kilpper -Balz based on genomic analysis [3].
Most common clinical infections caused by Enterococci include urinary tract infections (UTI"s), bacterial endocarditis, bacteremia, diverticulitis, wound infections, intra-abdominal infections, catheter related infections, surgical wound infections, pelvic infections and meningitis, respiratory infections, pleural space infections, skin and soft tissue infections have also been reported [4]. Till date around 36 Enterococcal species have been identified and 26 are associated with human infections [5][6][7]. Enterococcus faecalis is the most common human pathogen (70-90%) followed by enterococcus faecium (5-15%) that has been increasingly prevalent in hospital acquired infections.
Enterococci are emerging as one of the commonest organisms causing nosocomial infections in recent decades [9]. This is attributed by biofilms with multiple antibiotic resistance due to microbial characteristics like such as virulence factors and resistance genes that are magnified by the usage of contamination diagnostic and surgical procedures among the people [8][9][10]. Enterococcus has both the intrinsic and acquired resistance to many antibiotics that make them an important nosocomial pathogen. Enterococci show intrinsic resistance to cephalosporins, Blactams, aminoglycosides, lincosamides, Enterococci express low affinity to penicillin binding proteins (PBP"s) that bind weakly to βlactam antibiotics. For E. faecium PBP5 and for E. faecalis PBP4. As a result, minimum inhibitory concentrations (MICs) for penicillins are 2 -8 mg/ml for E. faecalis and 8-16 mg/ml for E. faecium [11].
Thus the accretion and spread of antibiotic resistance determinants among Enterococci, to the point where some clinical isolates that are resistant to all standard therapies, highlight both the vulnerability of our present armament as well as the looming prospect of a "post-antibiotic era". Thus, it results in increase in the cost burden for the healthcare. And it is also responsible to increase the morbidity and mortality. So efforts must be taken to prevent the emerging antibiotic resistance and spread of Enterococcal infections through infection control and stewardship programs implemented in the hospitals as well as in the community [12,13].
So, in context with the above perspective, this study was carried out in Sree Balaji Medical College and Hospital, Chennai.

METHODS
Prospective study: This study was conducted in the Department of Microbiology and central laboratory at the Sree Balaji Medical College And Hospital, Chrompet. Chennai.

Study Population
The study population included patients in different treating units from both outpatient department and inpatient department attending Sree Balaji Medical College and Hospital during the study period.

Study Period
The study was conducted for a period of one year from December 2015 to December 2016.

Statistics
The statistical analysis was performed using Microsoft Word 2010 and Microsoft Excel 2010 for entering data, creating tables and charts. Reports were expressed in percentage and frequency.

METHODOLOGY
A total of 17512 heterogeneous clinical samples that is urine, pus, blood, tissue fluids and ear swab taken from both inpatient and outpatient departments, from all the age groups, of both sexes submitted to the Central laboratory, Microbiology laboratory, of Sree Balaji Medical College And Hospital, for a period of one year were analyzed. A total of 190 Enterococcus isolates were recovered and further speciation and antibiotic susceptibility pattern was done.

RESULTS
A total of 17512 samples were analyzed for the prevalence of Enterococcus and speciation and antibiotic sensitivity was done after the identification. Further the age-group and sex wise distribution of Enterococcus commonly involved are identified.
Out of a total of 17512 heterogeneous samples obtained majority were obtained from inpatients department. 13792 (78.8%) samples were from inpatients and 3720 (21.2%) from the outpatients.
Out of a total of 17512 specimen were obtained that included 7953 of urine specimen, 5441 of pus samples, 3423 of blood samples, 695 of the exudates that included ear discharge, ascitic fluid and pleural fluid. The specimen like sputum, stool and vaginal swab were Enterococcus is a normal commensal were excluded from the study. The common Enterococcal species responsible for the infection and the other etiological factors related to the Enterococcal infections were identifid.
So according to this study the prevalence of Enterococcus among in-patients is 1.06% and outpatients is 1.1% (Chart 1). And the total prevalence of Enterococcus is 1.08%. In our study among 190 isolated Enterococcal samples 67 (35.2%) were collected from the Medicine department, 46 (24.2%) from surgery  So the overall prevalence of Enterococcus in Intensive care unit at SBMCH is 8.4% of the total enterococcal isolates and 0.9% of the total isolated organisms ( Table 3).
And it is less prevalent in children up to 12 years of age (7.8%) then in adults (92.1 %) of the total isolates (Table 5).
So, the child and adult ratio is 1:11.6 (Chart 3).   Since all the isolates showed sensitivity to vancomycin with MIC ranging from 0.5 -4 ug/ml by both the methods, Henceforth they are interpreted as vancomycin sensitive as per CLSI guidelines (Table 7).

DISCUSSION
Enterococci is distributed widely in nature and normally it constitutes a part of mixed flora in the gastrointestinal tract so it is difficult to differentiate the pathogen from the normal colonization [14]. Enterococci are one of the leading cause of nosocomial infections and opportunistic infections in the immune compromised patients worldwide that is attributed to the intensive use of broad spectrum antimicrobial agents [8,[15][16][17]. The various life threatening infections caused by Enterococci include bacteremia, endocarditis, peritonitis, urinary tract infections, wound infections and device related infections [10]. It possesses specific traits that enable the organism to survive in the adverse environmental conditions including the hospital conditions. Any isolate that is suspected to be Enterococcus is a gram -  [18]. Serious Enterococcal infections are difficult to treat as it has tremendous ability to acquire resistance to penicillin, high level aminoglycosides and glycopeptide resistance. So, it possess a great challenge for the clinicians and health care institutions as the multidrug resistance complicates the treatment and therapeutic spectrum becomes limited [19]. In this context our study aimed to detect the prevalence, conventional methods of isolation, identification and speciation, done by using the conventional tests proposed by R.R.Facklam and Collins, enterococci in di fferent clinical isolates along with the in vitro susceptibility testing [5]. In our study a total of 190 enterococci were isolated from a total of 17512 heterogeneous clinical samples both from outpatient and inpatient departments. The total of 21.2% Enterococcal isolates were obtained from outpatient department with a prevalence of 1.1% while 78.8% of the total isolates were obtained from inpatient department and a prevalence of 1.06%. As majority of specimen were obtained from inpatients. The results are similar to the findings of Acharya et al. who has reported 28% from outpatients and 72% among inpatients [20].

ENTEROCOCCAL SPECIES
According to our study, age wise distribution showed maximum number of isolates were obtained from the ag e group 40-59 years of age (35.7 %). Which differs from the studies by Preeti et al that showed highest prevalence of 40% between 21-40 years of age followed by 0 -20 and according to the study by Saroj et al and Parameswarappa et al that showed its predominant ≥61 years of age. Majority of the cases were isolated from adults patients 92.1% and only 7.8% from pediatric patients that does not resemble the study by Acharaya et al who has isolated 30.5% of enterococci from pediatric patients [21][22][23].
And from ICU a total of 8.4% of enterococci were isolated. The study differs from Salem Bekhit MM. et al that has reported 85% of isolates from ICU. And corresponds to the study by Paule et al who have reported only13.9% of the enterococcal isolates from ICU [24]. As per our study on the antibiotic susceptibility pattern we found that most of the Enterococcal isolates are highly resistant to penicillin 86.8%. The resistance among E. faecalis is 90% and that of E. faecium is 95.7%. This suggests that E. faecium is highly resistant to penicillin. The finding is consistent with the reports from Gordon et al. [6]. Resistance [24].
Since the identification and susceptibility are detected within 3 -6 hours by Vitek 2 System it appears as the most reliable and fast method and similar results as that of E-test were obtained after the isolates were subjected to Vitek 2 System that is 100% sensitivity to vancomycin and teicoplanin. This is comparable to the study by Nicole Van den Braak et al the overall agreement of vancomycin susceptibility testing with the Vitek 2 compact system (biomeriux) compared with the reference agar dilution method detecting MIC was 94% (184 of 195) and the overall agreement of the teicoplanin testing results between the two methods was 97% [26]. However vancomycin resistant strains have been increasingly reported worldwide but in our study no such resistant strains were isolated. So, the judicious use of these antimicrobials is recommended for the multidrug resistant isolates of Enterococci in order to restrict the emergence of resistance to these drugs.

CONCLUSION
Enterococcal infections are emerging as the significant pathogen responsible for both nosocomial infections as well as community acquired infections. There is an immense increasing multidrug resistance among Enterococci that contributes significantly to the mortality and mortality among the patients. There has been a change in species isolated from various clinical specimens as more of the E. faecium is isolated that suggests the change in the pattern of the infections caused by Enterococci. According to the study E. faecium is highly multidrug resistant and this suggests the species level identification of the Enterococcal isolates is important to treat the Enterococcal infections as well as to limit the emerging resistance as it can predict the patterns of antimicrobial susceptibility. There is a stringent need for prevention and control of spread of multidrug resistant Enterococci that could be achieved by the vigilant use of antimicrobial agents, close monitoring of higher drugs like vancomycin with its minimum use and looking for patients compliance, early detection and reporting by the laboratories, antibiotic prescribing policy and the audit, educating and screening of the healthcare workers and hospital staff, maintaining the corrective reflexive practices such as proper hand washing technique and immediate implementation of the appropriate infection control measures that will reduce the mortality and morbidity that can further prevent the organism to become a formidable clinical problem in the years to come.

CONSENT
As per international standard or university standard, patients' written consent has been collected and preserved by the author(s).

ETHICAL APPROVAL
The study is approved by the institutional ethical committee, Sree Balaji Medical College and Hospital.