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Study of Antimicrobial Resistance in Enterococci at Government Medical College, Bhavnagar, Gujarat, India

ISSN: 2319-7706 Volume 5 Number 9 (2016) pp. 826-836 Journal homepage: http://www.ijcmas.com Enterococci were originally classified as enteric gram-positive cocci and later, included in the genus streptococcus. The intrinsic and acquired antimicrobial resistance properties of Enterococci, to several antibiotics, have enabled them to survive in clinical environment Enterococci acquire resistance to several available antimicrobial agents by either mutation or by receiving the foreign resistant determinations through plasmids & transposons. The Aim of this research work is to study antimicrobial resistance in Enterococci . The present prospective study was conducted on 125 pure isolates of Enterococci isolated consecutively from various clinical samples like Pus, Blood, wound Swab, Sputum, urine, etc. Received at Department of Microbiology of Govt Medical College, Bhavnagar for bacteriological culture and sensitivity. The samples obtained were processed for culture of the bacteria as per routine standards methods. Detection of VRE and VSE is done. Chisquare was used to compare differences in resistance to antibiotics among the entercoccal species. A þ value of <0.05 was used to indicate significant differences. 22 (2.3%) pure enterococcal isolates were recovered from 921 specimens. The most frequent source of enterococcal isolations in this study was urine (63.63%) and greater rate of isolation of Enterococci from patients admited in wards (88.80%) as compared to isolates from outdoor patients Overall, this study revealed E.faecalis as the most common species (71.42%) followed by E.faecium (28.57%).The isolates were resistant to Penicillin (43.75%), Ampicillin (37.5%), Gentamicin (50%), Erythromycin (96.87%), Tetracycline (28.1%), Ciprofloxacin (75%). None of the isolates were resistant to linezolid. Two (0.64%) strains were resistant to vancomycin and Teicoplanin. All the strains (100%) in this study were resistant to Erythromycin. It was reassuring that 98.73% and 77.21% of the E.faecalis and 97.67% and 13.95% of the E.faecium in this study were vancomycin and Ampicillin susceptible, respectively. None of the isolate was resistant to linezolid and Tetracycline resistance was found only in 20.00% of isolates, suggesting their possible role in VRE and multi-drug resistant infection. The most frequent source of enterococcal isolations in this study was urine (63.63%) and greater rate of isolation of Enterococci from patients admitted in wards (88.80%) highlights the organisms as one of the important cause of nosocomial urinary tract infections. Overall, in the present study, the isolates were resistant to Penicillin (43.75%), Ampicillin (37.5%), Gentamicin (50%), Erythromycin (96.87%), Tetracycline (28.1%), Ciprofloxacin (75%). None of the isolates were resistant to linezolid. Two (0.64%) strains were resistant to vancomycin and Teicoplanin. K e y w o r d s Enterococci, Resistance, VRE and VSE Gujarat. Accepted: 25 August 2016 Available Online: 10 September 2016 Article Info Int.J.Curr.Microbiol.App.Sci (2016) 5(9): 826-836


Introduction
Enterococci are catalase negative gram positive cocci that occur singly or arranged in pairs or as short chains. They are ubiquitous in nature. Enterococci are traditionally regarded as low grade pathogens but have emerged as an increasingly important cause of nosocomial infections in the 1990s The ability of enterococi to have intrinsic resistance as well as to acquire resistance to several classes of antibiotics enhances their importance as human pathogen, especially in the nosocomial setting (Arthur et al., 1993).
The term 'enterococcus' probably originated with the discovery of the first organism of this group Thiercelin (1899) used this term to describe bacteria seen in pairs and short chains in human faeces. The name Streptococcus faecalis was used by Anderws and Horder (1906) (Agarwal et al., 1999) to identify an organism of faecal origin that clotted milk and fermented mannitol and lactose but not raffinose. Orla Jensen (1919) described a second organism, S.faecium which differed from the fermentation patterns of S-faecalis (Orla Jensen et al., 2005).
Murray BE (1990) reported that Enterococci were originally classified as enteric gram positive cocci and later included in the genus streptococcus (Murray, 1990). Lancefield R.C (J Exp Med 1933) traces that in the early 1930's Enterococci were classified as group D steptococci and were differentiated from the non-enterococcal Group D streptococci by distinitive biochemical characteristics (Lancefield, 1933). Sherman JM (1938) recommended that the term enterococcal group be specifically used for the streptococci that grow both at 10 0 C and 45 0 C at pH 9.6,in the presence of 6.5% NaCl, survive at 60 0 C for 30 minutes and hydrolyseesculin (Sherman, 1937).

Materials and Methods
The present prospective study was conducted on 125 pure isolates (

a)
Vancomycin disc diffusion method using vancomycin (30ug) disc susceptibility testing by modified Kirby -Bauer disc diffusion method.
b) Vancomycin agar screen method -(vancomycin 6 ug/ml) was used for true detetion of vancomycin resistance.
5. Statistical Analysis: Chi-square was used to compare differences in resistance to antibiotics among the entercoccal species. A þ value of <0.05 was used to indicate significant differences.
Detection of VRE and VSE is done. Chisquare was used to compare differences in resistance to antibiotics among the entercoccal species. A þ value of <0.05 was used to indicate significant differences.

Culture of the specimen & identification of genus entercoccus
Isolates received from clinical samples were presumptively identified as Enterococci by colony morphology. Morphology on Gram's staining, the absence of catalase production, the presence of pyrrolidonyl arylamidase by hydrolysis of L-pyrrolidonyl -Bnaphthylamide (Himedia Labs),tolerance to 65% sodium chloride.

(A)
Clinical samples like urine, Pus, Body fluids, etc were inoculated on the following media: Mac Conckey Agar (MCA) c) Blood was inoculated into blood culture bottle containing brain heart infusion broth.
(B) Culture media after inoculation were incubated at 35-37 0 C for 18-24 hrs aerobically in an incubator.
(C) Media were examined for microbial growth.
(D) Identification of the colony as enterococcus was done on the basis of: a) Colony characteristics. b) Morphology on Gram's staining c) Catalase test. d) Tolerance to bile esculin. e) Salt (6.5 % Sodium Chloride) Tolerance test. f) PYR Test (Hydrolysis of L -Pyrrolidonylßnapthylamide).

Anti-microbial susceptibility testing of of Enterococcal Isolates
All the Enterococcal isolates were subjected to modified Kirby-Bauer disc diffusion susceptibility using standard techniques as per (CLSI 2009) recommendations.
Antibiotic susceptibility testing was done by Kirby-Bauer disk diffusion method using antibiotic discs and Muller Hinton Agar (Himedia) as recommended by CLSI.
The antibiotic discs and their potency (antibiotic content) that was used for susceptibility testing of enterococcal isolates is listed below: After placing the antibiotic discs on the agar surface, the plates were incubated at 37 0 C for 18-24 hrs.
After 18 -24 hrs of incubation, the plates were viewed with unaided eye using reflected light for the presence or absence of zones of inhibition around each of antibiotic discs.
If present, the zone of inhibition around the respective antibiotics discs was measured using a ruler to within the nearest millimeters, including the diameter of the disc.

Modified Kirby: Bauer disc diffusion Susceptibilty testing with Standard strain of Staphylococcus aureus-ATCC 25923
Modified Kirby Bauer disc diffusion Susceptibility testing with Enterococcal strain

Result and Discussion
The zones of inhibition obtained by measurement were interpreted by comparison with reference zone sizes for the respective antibiotic disc when tested for

Detection of Vancomycin Resistance in Enterococci
Vancomycin was used for detection of vancomycin resistance.

Using 30 ug vancomycin disc in antimicrobial susceptibility testing by modified Kirby-Bauer disk diffusion method
After 24 hr of incubation, the Muller Hinton agar plate containing the vancomycin disc was examined with unaided eye using transmitted light for presence or absence of inhibition zone around the disc. Inhibition zones, if present, were measured with ruler. Any visible growth within the inhibition zones was also noted.     The distribution of isolates among all the clinical specimens is given in Table No.6. Of all the 22 enterococcal isolates 14 (63.63%) strains were isolated from Urine, 05(22%) from Pus and Wound swabs and 02(9%) from Blood and 1(4.5%) from lower respiratory tract (Table -6) E.faecalis 16(68.75%) was the most common species isolated from the clinical samples followed by E.faecium 06(21.87%).
No enterococcal isolate was recovered from body fluids and CSF.    The wider spread use of glycopeptides in hospitals has lead to the emergence of vancomycin resistant enterocooci (VRE), which is a major concern for health care professionals. Treatment of infections caused by VRE is a challenging task especially because the resistance appears in strains, which are multi-resistant. The optimal therapy for such infections is not known. Thus, acquired resistance to vancomycin by Enterococci greatly reduces the number of treatment options for disease management and the problem is further compounded by the fact that resistance genes can potentially be transferred to toher pathogenic organisms, such as staphylococcus aureus and streptococcus species (Carias et al., 1998).
Thus, measures should be taken to prevent further development and transmission of these infections by strictly implementing infection control guidelines and antibiotic policies in hospitals. Prudent use of antibiotics and a proper surveillance for VRE may permit early recognition and containment of spread of this emergency pathogen in our country (Center for Disease control and Prevention, 1989).
Moreover it has become more difficult for treating physicians to treat such multi -resistant enterococcal strains due to the lack of adequate information regarding the species specific anti-microbial resistance pattern worldwide. There is also a paucity of information on species specific antimicrobial resistance pattern in Enterococci from our country.
Thus, looking to the impending need for constant monitoring of the species prevalence and antimicrobial resistance pattern (including VRE & VSE) of local enterococcal strains and its epidemiology, the present study was conducted in our setting. The present study results are consistent with other studies conducted elsewhere in India and abroad (Boyce et al., 2004). Mathur et al., (2003) reported 66% Enterococci to be ampicillin resistant which is in accordance to the present study result. From India, Karmarkar et al., (2004) reported 100% E.faecalis and 85.7% E. faecium to be resistant to this drug. This incidence is much lower than that obtained in the present study but much higher than that obtained in another study from Delhi (Mathur et al., 2003) in which they reported only 26% E. Faecalis strains to be gentamicin resistant.
The incidence of VRE in the present study is 0.64%, which reflects the emergence of VRE in Govt medical college, Bhavnagar, Gujarat. Because of the limited therapeutic options for treating serious infections caused by VRE; it has emerged as one of the leading clinical challenge for physicians.
In conclusion, the most frequent source of enterococcal isolations in this study was urine (63.20%) and greater rate of isolation of Enterococci from patients admitted in wards (88.80%) highlights the organisms as one of the important cause of nosocomial urinary tract infections.Overall, in the present study, the isolates were resistant to Penicillin (43.75%), Ampicillin (37.5%), Gentamicin (50%), Erythromycin (96.87%), Tetracycline (28.1%), Ciprofloxacin (75%). None of the isolates were resistant to linezolid. Two (1.60%) strains were resistant to vancomycin and Teicoplanin.