Antimicrobial Susceptibility Patterns of Common and Unusual Enterococcus species Isolated from Clinical Specimens

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1 Original Article Vol. 24 No. 2 Isolation and sensitivity pattern of enterococci:- Chaudhary U, et al. 55 Antimicrobial Susceptibility Patterns of Common and Unusual Enterococcus species Isolated from Clinical Specimens Uma Chaudhary, M.D. (Microbiology)* Madhu Shamma, M.D. (Microbiology)** Aparana Yadav, M.D. (Microbiology)*** ABSTRACT Multidrug-resistant enterococci are emerging as a leading nosocomial pathogen. Knowledge of the antimicrobial resistance profile is essential to formulate treatment guidelines for infections caused by enterococci. A total of 260 enterococcal strains were isolated from various clinical specimens between March 2004 and December 2005 at the Department of Microbiology, Post Graduate Institute of Medical Sciences, Rohtak, Haryana, India. They were speciated, and an antimicrobial susceptibility pattern was determined in 72 percent of strains isolated. E. faecalis was the most common species in all clinical specimens except in blood cultures in which E. faecium was more common (50%). Polymicrobial infection was noted in 46 (17%) of the isolates. The maximum susceptibility was observed with vancomycin (98%), followed by teicoplanin (88%), linezolid (79%), novobiocin (78%), spectinomycin (55%), and doxycycline (51%). Obtaining effective control of multidrug-resistant enterococci would require prudent antibiotic usage, better isolation procedures in hospital and other patient care environments, and also improved and rapid surveillance measures. (J Infect Dis Antimicrob Agents 2007;24:55-62.) INTRODUCTION Recent years have witnessed an increasing interest in enterococci not only because of their ability to cause serious infections like endocarditis, bacteremia, and intraabdominal infections but also because of increasing resistance to many antimicrobial agents. 1 Risk factors for developing enterococcal infections are prolonged hospitalization especially in intensive care units (ICUs), surgical procedures following liver transplants, immunocompromised states, breakdown of normal physical barriers (gastrointestinal, skin, and urinary tract), and neurosurgical procedures. *Senior Professor & Head, M.D. (Microbiology), Department of Microbiology, PGIMS,Rohtak. **Senior Resident, M.D. (Microbiology), Department of Microbiology, PGIMER Chandigarh. ***Associate Professor, M.D. (Microbiology), Department of Microbiology, PGIMS, Rohtak. Received for publication: April 20, Reprint request: Dr. Aparna, Associate Professor, Department of Microbiology, Pt. B.D. Sharma, PGIMS, Rohtak shamataluja@yahoo.com Keywords: Enterococcus, vancomycin-resistant enterococci, aminoglycoside resistance, susceptibility 55

2 56 J INFECT DIS ANTIMICROB AGENTS May-Aug Enterococcus faecalis causes percent of human enterococcal infections while E. faecium accounts for majority of the remainder. Other Enterococcus spp. including E. avium, E. casseliflavus, E. durans, E. gallinarum, E. hirae, E. malodoratus, E. mundtii, E. raffinosus, and E. solitarius are infrequent causes of human infections. 2 Enterococci are intrinsically resistant to many antimicrobial agents including aminoglycosides, clindamycin, antistaphylococcal penicillins (oxacillin, methicillin, and nafcillin), cephalosporins, and most fluoroquinones. 3 A combination of penicillin and gentamicin has been the mainstay of the treatment of enterococcal infections until recently but with the emergence of high-level aminoglycoside resistance (HLAR), vancomycin is now the only alternative available. The widespread use of glycopeptides in hospitals has led to the emergence of vancomycinresistant enterococci (VRE) which is a now a major concern for health care professionals. Knowledge of the profile of antimicrobial resistance is essential to formulate the treatment guidelines for infections caused by enterococci. This study reports the isolation and antimicrobial susceptibility of common and unusual Enterococcus species isolated from clinical specimens of patients admitted to a tertiary care hospital in Haryana, India. MATERIALS AND METHODS The study was conducted in the Microbiology Department of Post Graduate Institute of Medical Sciences, Rohtak, Haryana, India. A retrospective study was conducted over a period from March 2004 to December 2005 in which 260 strains of enterococci were isolated from various clinical specimens (N=1445). The analyzed specimens originated from the urine, pus, blood, sputum, throat swab, stool, cerebrospinal fluid (CSF), and body fluids. The specimens were plated on blood and McConkey agar plates which were incubated at 37 C for hours. Enterococci were identified and speciated by using conventional test scheme suggested by Facklam. 4 All isolates identified as enterococci were tested for antimicrobial susceptibility by the modified Stoke s disc diffusion method using reference strain, E. faecalis NCTC Inocula were prepared from overnight growth on a blood agar plate by suspending seven to eight morphologically similar colonies in glucose broth. Each inoculum was adjusted to 0.5 McFarland standard. The antibiotic susceptibility was carried out on brain heart infusion agar (Hi-media Mumbai, India). All strains were tested for susceptibility using penicillin (10 µg), ampicillin (10 µg), imipenem (10 µg), meropenem (10 µg), vancomycin (30 µg), teicoplanin (30 µg), gentamicin (120 µg), rifampicin (5 µg), norfloxacin (10 µg), doxycycline (30 µg), tetracycline (30 µg), erythromycin (15 µg), chloramphenicol (30 µg), nitrofurantoin for urine isolates (300 µg), spectinomycin (100 µg), novobiocin (30 µg), and linezolid (30 µg) disks. The minimum inhibitory concentration (MIC) was determined by using the standard agar dilution method for all the antibiotics except vancomycin and gentamicin for which agar screening method was used. 6 RESULTS A total of 260 enterococci were isolated from various clinical specimens (N=1,445) during the study period. Based on the biochemical reactions, only 234 enterococci could be speciated. E. faecalis (72.3%) was the most common species isolated in all clinical specimens except in blood cultures where E. faecium (50%) was the most common isolate (Table 1). Twelve enterococci showed atypical reactions including 5 mannitol-negative variant of E. faecalis-like species, 3 arginine-negative E. faecalis-like species, 3 mannitol-

3 Vol. 24 No. 2 Isolation and sensitivity pattern of enterococci:- Chaudhary U, et al. 57 negative variant of E. faecium like species, and 1 arginine-negative E. casseliflavus-like species. These enterococci were isolated from clinical specimens including the pus, urine, blood CSF, stool, and vaginal swabs (Table 2). The pus specimens were obtained from patients with burn wound and empyema. Urine specimens were obtained from patients with polyarthritis, urinary tract infection, sepsis with uterine perforation, vesico-vaginal fistula, tuberculosis with urinary tract infection, while blood specimens were obtained from patients with neonatal jaundice, empyema, tuberculosis, sepsis with uterine perforation, cirrhosis, hepatitis, Tetralogy of Fallot with suspected brain abscess, and rheumatic heart disease. CSF specimens were obtained from patients with tuberculosis and Tetralogy of Fallot. Other 16 enterococci were isolated from stool specimens and vaginal swabs. The infection was polymicrobial in 46 (17.7%) patients. Along with enterococci, other organisms including Escherichia coli, Klebsiella, Enterobacter, Citrobacter, Acinetobacter, coagulase-negative Staphylococcus, Pseudomonas aeruginosa, and Candida spp. were also isolated (Table 3). Table 4 shows the susceptibility patterns of various species of enterococci to different antimicrobial agents. Maximum subceptibility was observed with vancomycin (98.1%), followed by teicoplanin (88.57%), linezolid (79.44%), novobiocin (78.94%), spectinomycin (55%), imipenem (53.3%), and doxycycline (51%). The susceptibility to other drugs was very low. All the species were maximally susceptible to vancomycin except for E. casseliflavus which showed maximum susceptibility to linezolid. DISCUSSION Despite the fact that enterococci have been considered to be relatively low virulent, in the past few years these organisms, among all nosocomial pathogens, have emerged as a significant concern. Data indicates that incidence of nosocomial enterococcal infections have been increasing, and enterococci have become the second leading cause of nosocomial infections. 7-9 Various species isolated in our study were E. faecalis (72.3%), E. faecium (17.3%), E. raffinosus (2.3%), E. durans (2.3%), E. casseliflavus (1.92%), Table 1. Distribution of Enterococcus species in 1,445 clinical specimens. Enterococcus species (number) Specimens (number, %) Urine Blood Pus CSF Stool and body fluids Throat swab and sputum E. faecalis 180 (72.3%) 40 (82%) 20 (40%) 36 (72%) 34 (68%) 26 (86.64%) 24 (93.33%) E. faecium 45 (17.30%) 3 (6%) 25 (50%) 4 (8%) 2 (4%) 4 (13.33%) 1 (3.33%) E. raffinosus 8 (2.3%) 2 (4%) 2 (4%) 2 (4%) 2 (4%) - - E. durans 6 (2.3%) - 1 (2%) 2 (4%) 3 (6%) - - E. casseliflavus 5 (1.92%) 1 (2%) - 1 (2%) 3 (6%) - - E. dispar 4 (1.53%) 1 (2%) 1 (2%) 1 (2%) 1 (2%) - - Unspeciated 12 (4.6%) 2 (4%) 1 (2%) 4 (8%) 5 (10%) - 1 (3.33%) Total isolates 260 (100%) CSF: cerebrospinal fluid 57

4 58 J INFECT DIS ANTIMICROB AGENTS May-Aug Table 2. The prevalence of enterococci in clinical specimens in various clinical conditions. Clinical conditions Clinical specimens Number Number of enterococci isolated Burns Pus Urine Polyarthritis with UTI Urine UTI Urine Neonatal jaundice Blood Empyema Pus Tuberculosis with/without UTI Blood 40 4 Urine CSF 50 3 Sepsis with uterine perforation Urine Blood 60 2 Cirrhosis Blood Hepatitis Blood Tetralogy of fallot with brain abscess Blood 20 5 CSF 20 2 Vesico-vaginal fistula Urine 32 5 Rheumatic heart disease with severe MS Blood 28 3 Diarrhoea Stool Vaginitis Vaginal swab 15 6 MS: mitral stenosis, CSF: cerebrospinal fluid, UTI: urinary tract infection Table 3. The prevalence of concomitant organisms along with enterococci in various clinical specimens. Organism Blood Pus Urine CSF and Vaginal Stool body fluids swabs Escherichia coli Klebsiella spp Enterobacter spp Citrobacter spp Acinetobacter spp Coagulase-negative Staphylococcus spp Pseudomonas aeruginosa Viridans Candida spp CSF: cerebrospinal fluid

5 Vol. 24 No. 2 Isolation and sensitivity pattern of enterococci:- Chaudhary U, et al. 59 Table 4. The susceptibility of various-species of enterococci. Antibiotic Total E. faecalis E. faecium E. casseliflavus E. raffinosis E. dispar E. durans (number, %) Penicillin Imipenem Meropenem Vancomycin Teicoplanin Gentamicin Doxycycline Tetracycline Erythromycin Chloramphenicol Norfloxacin Nitrofurantoin* Rifampicin Spectinomycin Novobiocin Linezolid *Urine isolates and E. dispar (1.53%). Udo and colleagues isolated 415 enterococci during a clinical study. In their study, there were E. faecalis (85.3%), E. faecium (7.7%), E. casseliflavus (4.0%), E. avium (1.2%), E. durans (1.0%), E. gallinarum (0.5%), and E. bovis (0.2%). 10 Ghoshal and colleagues isolated 685 enterococci from clinical specimens. Of them, 67 percent were E. faecalis, and 33 percent were E. faecium. 11 In our study, E. faecalis outnumbered other enterococci in all clinical specimens except in blood cultures in which E. faecium (50%) outnumbered E. faecalis (40%) and other enterococci which is in concordance to the study of Mohanty and colleagues. 12 Enterococci isolated from various clinical specimens in our study probably do not reflect the true 59 incidence of infections caused by this organism but definitely suggest the increased frequency of their isolation. In our study, the prevalence rate of enterococci was 18 percent, compared with 22 percent in a study of urinary pathogens by Desai and collegues. 1 We found the highest prevalence of enterococci isolated from burn patients (26.9%), followed by those with polyarthritis with urinary tract infection (15.38%). Although wound colonization was high in burn patients, none had sepsis due to enterococci. Desai and collegues observed that the most frequent sources of isolated enterococci were Foley s catheters (48.21%) and burn wounds (29.51%). 1 The unusual Enterococcus species including

6 60 J INFECT DIS ANTIMICROB AGENTS May-Aug E. raffinosus, E. durans, E. casseliflavus, and E. dispar were mostly isolated from the urine, blood, pus, CSF, and body fluids. A study from South India has also shown that the most common source of enterococcal isolations were the urine, blood, and burn wound. 13 Several studies have demonstrated that E. gallinarum and E. casseliflavus colonize the gastrointestinal tracts of both hospitalized patients and non-hospitalized healthy individuals. 14,15 We could not isolate any E. gallinarum in our clinical specimens. Enterococci are usually part of a mixed flora commonly found in the gastrointestinal tract and it remains difficult to differentiate colonization from true infection. Interactions among various bacteria have been demonstrated, and several studies suggest that enterococci can act synergistically with other intestinal bacteria to enhance the rate of infection. 14 In our study, the enterococcal infection was polymicrobial in 46 (17.7%) patients (Table 2). In a study from South India by Prakash and colleagues, 13 percent of enterococcal infections were polymicrobial. 12 Vancomycin usage has increased in hospitals following the emergency of methicillin-resistant Staphylococcus aureus. The emergence of VRE is also due to the inappropriate use of cephalosporin as well as poor hospital infection control measures. 16,17 Isolates of E. faecium were found in our study to be more multi-resistant than E. faecalis. E. faecium isolates were significantly more resistant to spectinomycin, nitrofurantoin, penicillin, tetracycline, and meropenem. However, the meropenem susceptibility should be interpreted with caution since the Clinical Laboratoty Standards Institute recommends only ampicillin disk diffusion method to determine imipenem susceptibility providing the species is confirmed to be E. faecalis. Dubre and colleagues reported that E. faecium isolates exhibited a higher percentage of resistance to penicillin, erythromycin, and ampicillin while E. faecalis isolates were more resistant to tetracycline and penicillin. 18 Almeida and colleagues reported a higher percentage of resistance in E. faecalis to ampicillin, penicillin, aminoglycosides (highlevel resistance), chloramphenicol, ciprofloxacin, rifampicin, and erythromycin while E. faecium isolates exhibited more resistance to tetracyclines. 19 We isolated 5 VRE with MIC of 32 µg/ml. Of these, 2 were E. faecalis, and 3 E. faecium. 4 VRE were isolated from the stool, and 1 vancomycin-resistant E. faecium was isolated from the blood. The emergence of VRE is a cause of concern because of the limited therapeutic options for treating serious infections and because of their potential to transfer vancomycin resistance genes to other organisms such as methicillin-resistant S. aureus. Conjugative in vitro transfer of resistance genes to S. aureus, Listeria monocytogenes, and group A and viridans streptococci have been reported. 20 Regarding the unusual species of Enterococcus, the susceptibility patterns did not show any major differences except for E. casseliflavus which showed 100-percent susceptibility to linezolid. These unusual species showed better susceptibility towards linezolid and novobiocin than E. faecalis and E. faecium. In conclusion, an identification of enterococcal isolates to the species level in the clinical microbiology laboratory is useful because it can help predict patterns of antimicrobial susceptibility. The emergence of glycopeptide resistance in this bacterial genus, especially in E. faecium, is of concern because of a very few antibiotics that are active against these bacteria. Moreover, since in certain strains, the resistance is mediated by self transferable plasmids with a broad-host range, one can confidently predict the dissemination of this new type of resistance to other Gram-positive human pathogens. 8 The appearance of plasmid-mediated transferable resistance to major antibiotic classes emphasizes, once more, not only on

7 Vol. 24 No. 2 Isolation and sensitivity pattern of enterococci:- Chaudhary U, et al. 61 the necessity for more discriminate use of new drugs but also for continuous efforts to find or design antimicrobial agents. Thus, we suggest intensified actions to promote more the rational use of antibiotics in health care settings, more surveillance studies in order to monitor changes in enterococcal resistance patterns and the adoption of measures to prevent the spreading of genetically related resistance isolates. References 1. Desai PJ, Pandit D, Mathur M, Gogati A. Prevalence, identification and distribution of various species of enterococci isolated from clinical specimens with special reference to urinary tract infection in catheterized patients. Ind J Med Microbiol 2001;19: Jett BD, Huycke MM, Gilmore MS. Virulence of enterococci. Clin Microbiol Rev 1994;7: French GL. Enterococci and vancomycin resistance. Clin Infect Dis 1998;27(Suppl 1):S Facklam RR, Collins MD. Identification of Enterococcus species isolated from human infections by a conventional test scheme. J Clin Microbiol 1989; 27: Agarwal KC. Antibiotic sensitivity test by disc diffusion method: standardization and interpretation. Indian J Pathol Bacteriol 1974;17: Clinical and Laboratory Standards Institute (CLSI). Performance standards for antimicrobial susceptibility testing; Sixteenth informational supplement. Vol. 26 No. 3. Approved standard M2-A9. Wayne, PA: CLSI, Schaberg DR, Culver DH, Gaynes RP. Major trends in the microbial etiology of nosocomial infection. Am J Med 1991;91(3B):72S-75S. 8. Murray BE. Diversity among multidrug-resistant enterococci. Emerg Infect Dis 1998;4: Nelson RR, McGregor KF, Brown AR, Amyes SG, Young H. Isolation and characterization of 61 glycopeptide-resistant enterococci from hospitalized patients over a 30-month period. J Clin Microbiol 2000;38: Udo EE, Al Sweih N, Phillips OA, Chugh TD. Species prevalence and antibacterial resistance of enterococci isolated in Kuwait hospitals. J Med Microbiol 2003;52: Ghoshal U, Garg A, Tiwari DP, Ayyagari A. Emerging vancomycin resistance in enterococci in India. Indian J Pathol Microbiol 2006;49: Mohanty S, Jose S, Singhal R, et al. Species prevalence and antimicrobial susceptibility of enterococci isolated in a tertiary care hospital of North India. Southeast Asian J Trop Med Public Health 2005;36: Prakash VP, Rao SR, Parija SC. Emergence of unusual species of enterococci causing infections, South India. BMC Infect Dis [online] 2005 [cited 2006 Jan 9];5(1):14. Available from: content/pdf/ pdf 14. Van Horn KG, Rodney KM. Colonization and microbiology of the motile enterococci in a patient population. Diagn Microbiol Infect Dis 1998;31: Gordts B, Van Landuyt H, Ieven M, Vandamme P, Goossens H. Vancomycin-resistant enterococci colonizing the intestinal tracts of hospitalized patients. J Clin Microbiol 1995;33: MacIntyre CR, Empson M, Boardman C, Sindhusake D, Lokan J, Brown GV. Risk factors for colonization with vancomycin-resistant enterococci in a Melbourne Hospital. Infect Control Hosp Epidemiol 2001;22: Shlaes DM. Vancomycin-resistant bacteria. Infect Control Hosp Epidemiol 1992;13: Dupre I, Zanetti S, Schito AM, Fadda G, Sechi LA. Incidence of virulence determinants in clinical Enterococcus faecium and Enterococcus faecalis isolates collected in Sardinia (Italy). J Med Microbiol

8 62 J INFECT DIS ANTIMICROB AGENTS May-Aug ;52: Titze-de-Almeida R, Rollo FM, Nogueira CA, et al. Molecular epidemiology and antimicrobial susceptibility of Enterococci recovered from Brazilian intensive care units. Braz J Infect Dis [online] 2004 [cited 2006 Jan 23];8(3): Available from: Noble WC, Virani Z, Cree RG. Co-transfer of vancomycin and other resistance genes from Enterococcus faecalis NCTC to Staphylococcus aureus. FEMS Microbiol Lett 1992;72:195-8.