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1Department of Microbiology, Father Muller Medical College, Mangalore, Karnataka, India
2Dr. Kuruvilla Thomas S, Professor, Department of Microbiology, Father Muller Medical College, Mangalore, Karnataka, India.
*Corresponding Author:
Dr. Kuruvilla Thomas S, Professor, Department of Microbiology, Father Muller Medical College, Mangalore, Karnataka, India., Email: thomssk@yahoo.comAbstract
Background: Enterococci are facultative anaerobic gram-positive cocci occurring in pairs or short chains, belonging to the family Enterococcaceae. They are catalase negative and are distributed widely in nature. There are many species of Enterococci that have been identified. The purpose of this study was to determine the frequency of different species of Enterococci from clinical samples with positive culture using conventional methods and to analyze their antibiogram to initiate appropriate therapy.
Methods: Out of a total 1200 clinical samples processed, 59 Enterococcal isolates were obtained from urine, pus, blood and wound swabs over a period of one year from all the patients admitted to our tertiary care centre. These samples were subjected to culture and sensitivity tests. All the suspected Enterococcal culture isolates were identified by conventional methods and their antibiogram was analyzed.
Results: The Enterococcal species commonly isolated in our study were E. feacalis and E. feacium. There was a predominance of E. faecalis 33 (55.9%) over E. faecium 26 (44.06) among the 59 Enterococcal infections. E. faecium was found to be multidrug resistant and sensitive only to Vancomycin.
Conclusion: There was a predominance of E. faecalis over E. faecium among the Enterococcal isolates. The multidrug resistant pattern of E. faecium warrants prompt identification and long term strategies to address the increasing frequency of antibiotic resistance among Enterococcal infections, particularly with reference to E. faecium, especially when empirical therapy must be initiated.
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Introduction
Enterococci are facultative anaerobic gram-positive cocci occurring in pairs or as a short chain of cocci.1 They belong to the family Enterococcaceae. They are catalase negative and are distributed widely in nature.2 They are categorized under phylum Firmicutes that comprises a wide genus of lactic acid bacteria. These ovoid cocci were previously known as faecal Strepto-cocci and were described as Group D Streptococcus until 1984. Later, the DNA analysis led to their categorization into a discrete genus.3 They are part of the commensal flora of the intestine and biliary ducts and are found in smaller numbers in the genital and urinary tracts. The most frequently isolated Enterococcus species causing human infections are Enterococcus faecalis, Enterococcus faecium, Enterococcus avium, Enterococcus malodoratous, Enterococcus gallinarum, Enterococcus cecorum, Enterococcus durans, Entero-coccus hirae, Enterococcus mundti, Enterococcus dispar and Enterococcus casseliflavus.4 Enterococci can survive extreme environments, including a high pH of 6.5% NaCl, 40% bile salts and temperatures ranging between 10ºC to 45ºC. Apart from being found in humans, they are also found in animals, birds, plants, insects, water, soil, food, etc. In humans, Enterococci can cause infections of the urinary tract, bacteremia, endocarditis, pelvic and intra-abdominal infections. They may also cause of wound, tissue and respiratory infections and meningitis.5
Materials and Methods
This prospective surveillance study was carried out after obtaining the institutional ethical committee clearance (FMIEC/CCM/365/2021) in the microbiology laboratory of a tertiary care hospital in Mangalore, for a period of one year. The samples, urine, pus, blood and wound swabs were obtained from all the patients admitted to the hospital and screened for Enterococal isolates. The samples received at the lab were processed immediately. Out of a total 1200 clinical samples processed, 59 (4.9%) Enterococcal isolates were isolated from the positive cultures.
A direct gram stain smear of the samples was done to observe Gram positive cocci in pairs, typically resembling Enterococci. The samples were then aseptically spread onto blood and MacConkey agar using a sterile nichrome wire loop. These petri dishes were incubated for 18-24 hours at 37ºC. Blood samples received in BACTEC blood culture bottles were loaded into the BACTEC 9120 equipment and the bottles that flagged positive were sub cultured. The morphological characteristics of the growth of suspected colonies on blood agar, including size, shape, color and hemolytic properties and growth of tiny deep pink magenta colored colonies on MacConkey’s agar were noted. The presumptive identification of these Enterococcal colonies was done by Gram stain, biochemical and sugar fermentation tests. The biochemical tests conducted were catalase, bile esculin, salt tolerance (6.5%) and heat tolerance tests (60ºC) for 30 minutes. Further speciation was done by the following tests: Voges-Proskauer (VP), motility test, arginine hydrolysis, tellurite reduction and sugar fermentation tests for lactose, mannitol, sorbitol, arabinose, raffinose and sucrose. The antimicrobial susceptibility testing by the Kirby Bauer disk diffusion method was then performed. The following antimicrobial agents were tested: Ampicillin (10 μg), High level Gentamicin (120 μg), Tetracycline (30 μg), Ciprofloxacin (5 μg), Erythromycin (15 μg), Linezolid (30 μg) and Vancomycin (30 μg). The susceptibility testing was quality controlled using ATCC strains of E. faecalis (ATCC 29212). The strains and antibiotic discs were obtained from Himedia Lab Pvt limited. Zone diameters were interpreted according to the standard guidelines. Statistical analysis was done by the percentage method.
Results
Among the 59 Enterococcal isolates from various samples isolated in the study, there was a predomi-nance of E. faecalis 33 (55.9%) over E. faecium 26 (44.06). No other species of Enterococcus was isolated in this study. 35 (55.3%) isolates were from males and 24 (40.7%) were from females. The isolates were from samples of patients of different age groups. There were six isolates from ≤ 20 years of age of which there was one neonate from whom E. faecalis was isolated from a blood culture sample. Twelve isolates were from the 21-40 years age group, 17 from the 41-60 years age group and 24 from above 60 years age group.
The distribution of the Enterococcal isolates from various samples is depicted in Table 1. Majority of the Enterococcal isolates were from urinary tract infections (33.7%), of which 10 (45.4%) were catheterized samples, pus aspirates (33.9%) from abscesses, blood cultures (18.6%) from cases admitted with fever and wound swabs from cases with skin and ear infections (10.2%). All the isolates were found to be catalase negative, tolerant to salt and heat and hydrolyzed esculin to esculitin. Inoculation on blood agar showed the presence of translucent colonies. All the isolates were non-motile and tested negative for VP test, hydrolyzed arginine and were positive for the Tellurite reduction test. The antibiogram of the isolates is depicted in Table 2.
Discussion
There are several reasons for the increased colonization of Enterococci among hospitalized patients, including prolonged stay in hospitals, empirical use of antibiotic medications, surgical and other invasive procedures, rectal thermometers, air fluidized microsphere beds, etc.6 In a study by Varghese et al., the species of Enterococci commonly involved in human infections were E. faecalis and E. faecium, which was consistent with the findings of our study.7 The identification and speciation of Enterococcal isolates play a major role in therapeutic choices since suscepti-bility of Enterococci to various antimicrobials differ between the species.1 Conventional methods may be cumbersome for routine work flow, while more expensive automated methods have their advantages. Antimicrobial susceptibility tests too can be done either manually, such as disc diffusion technique or by automated methods.1 The emergence of antibiotic-resistant strains also pose newer challenges to clinicians while treating severely infected patients. It limits the traditional therapeutic options and thus complicates the suffering of the patients. It may lead to prolonged hospitalization, increased treatment cost, treatment failure and death of the patients.8 These organisms have an ability to ferment sugars and produce acids, and the commercially available test kits are developed based on these characteristics.9 The role of Enterococci as one of the major causes of nosocomial infection has already been established. Increasing antibiotic resistance among Enterococci is now a major cause of concern. A predominance of E. feacalis over E. faecium was observed in our study and also a dominance of Enterococcal isolates in males. Most of the studies conducted by other researchers have also reported this trend. The increase may be attributed to increased outdoor activities, health and hygiene habits or co-morbidities among males. However, proliferation to large numbers in soil is unlikely due to their auxotrophies.10
Colonization of microorganisms in the newborn can occur through vaginal fluid, maternal intestinal commensals and mother’s milk.11 The increasing presence of Enterococci is attributed to their natural ability to acquire and share extra-chromosomal elements encoding virulence characteristics and antibacterial resistant genes.12 Colonization and biofilm formation are affected by extracellular surface proteins. It leads to adhesion to cells as demonstrated in endocarditis and urinary tract infections. In a study by Stevens et al., around 16% of E. faecium isolates from patients of nosocomial blood stream infections were found to be Vancomycin resistant.13 An inappropriate use of antibiotics during empirical medication for urinary tract infections have contributed to a significant increase in resistant strains.14 Their role as potential organisms in developing host immunity in neonates, especially during the first months of birth has been well identified. Breast feeding and contact with vaginal fluid during normal delivery may lead to the origin of Enterococci in the gastrointestinal tract of neonates.15 We had one case of neonatal septicemia and isolation of E. faecalis from blood culture, which was a central line associated blood stream infection and was a sensitive strain. Enterococci were found to have intrinsic resistance to antibiotics belonging to the cephalosporin and aminoglycoside groups. Thus a combination of penicillin with amino-glycosides is recommended. If the strains develop high level aminoglycoside resistance, this synergism is lost. Then the choice would be Vancomycin.16
The maximum number of Enterococcal isolates in our current study were from urine samples, followed by pus. This trend was similar to the reports of Shanmukhappa et al., and Vibi Varghese et al.6,7 Those older than 60 years were the most affected. This observation was similar to the findings of Shanmukhappa et al. The hemolytic property of Enterococcus does not provide any additional information on the pathogenic characteristics of Enterococci. In our study, 67.8% isolates were non-hemolytic. This is almost comparable to the findings of Shanmukhappa et al., who reported 63.75% of non-hemolytic isolates.6 A study by Ashfaq Ahmed Shah et al., observed 70% non-hemolytic isolates of Enterococci. The percentage of beta hemolytic isolates in our current study was 25.4%, while Shanmukhappa et al., reported 35%.6
E. faecium isolates in our study were found to be more resistant to multiple antibiotics and Shanmukhappa et al., also endorsed this fact. Current studies have reported Ciprofloxacin resistance among the isolates as 81.4%.6 This is consistent with our study where 75% ciprofloxacin resistance was observed among E. fecalis and 73% resistance among E. fecium isolates. The reported resistance among the Enterococcal isolates against Ciprofloxacin in a research work undertaken by Ameliwork Yielema et al., and Aasish Karna et al., were 70.8% and 61.5%, respectively.1,2 In our study, none of the isolates of E. feacalis exhibited Vancomycin resistance. This was similar to the observations of the research work by Shanmukhappa et al., and Paul M et al.4,6 The total percentage of Vancomycin resistant E. faecium isolates were 42.3% in our study. In India, the first report of Vancomycin resistant Enterococci (VRE) was published in 1999 from New Delhi. The report quoted the prevalence rate of VRE as 0.89%. Now it has risen to around 10%.7 The resistance to Vancomycin was seen only among strains of E. feacium in our study. These strains were isolated from four catheterized urine samples and they fulfilled the criteria for catheter associated urinary tract infection (CAUTI) and the two other VRE were isolated from pus samples. Similar observations were made by Suraj Shrestha et al., in their systematic review and meta analysis. VRE are a major cause of concern nowadays.3 VRE was first reported much earlier in 1986 from France and UK, but presently they are being witnessed all around the globe. Recent reports reveal an approximate 20-fold increase in these isolates over the past two decades.1 E. faecium vancomycin resistance is also on the rise.5
As vancomycin is the drug of choice in Enterococcal infections, the aspect of speciation is important.5 Resistance to Linezolid was not observed among our E. feacalis and E. feacium isolates. A research work by Subhender Sikadar et al., however showed lower rate of Linezolid resistance (1.23%).12 Among E. faecium isolates, the maximum resistance was seen towards Ciprofloxacin. Jyothi Parameswarappa et al., conducted a study including only urine samples collected from cases of urinary tract infections and reported multi drug resistance to be higher among E. faecium isolates compared to E. faecalis isolates.17 Among our isolates, drug resistance to Tetracycline and Erythromycin was found to be 26% each and the resistance to high level Gentamicin among E. faecium isolates was 80.7%, which was comparable with other studies.6 There was a favorable response to therapy among our patients based on the antibiogram of the isolates. Despite advances in automated identification methods, supplemented manual identification methods may also be required to correctly identify Enterococcal isolates to species level.18 Regardless of the method of speciation adopted in the laboratories, it is crucial to identify Enterococcal isolates to the species level so that caution is exercised for empirical use of reserve drugs. This information should be passed on to the clinicians as early as possible to prevent nosocomial dissemination of probable resistant Enterococci and thereby implement good antibiotic stewardship.
Conclusion
The current study illustrated a predominance of E. faecalis over E. faecium among Enterococcal infections. Although this was not statistically significant, a trend of increasing frequency of E. faecium infections has been noted worldwide. A rising proportion of Vancomycin resistant Enterococci reported globally is an eye opener to all involved in infection control. It is also essential to speciate Enterococci to raise awareness among the clinicians even as they proceed to treat suspected Enterococcal infections in healthcare facilities.
Conflict of Interest
Nil
Supporting File
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