Antibiogram Pattern and Virulence Trait Characterization of Enterococcus Species Clinical Isolates in Eastern India: A Recent Analysis

Introduction

Enterococci, normally considered commensal members of healthy intestinal microbiota of humans and animals, have gained widespread importance due to their increased isolation rates in both community-related and nosocomial infections with substantial morbidity and mortality.^[1,2] Worldwide, enterococci are considered the second most common etiologic agent of urinary tract infections and third of nosocomial bacteremia.^[1,3] Other significant infections caused by enterococci include peritonitis, cholecystitis, meningitis, wound, and soft tissue infections, catheter-related infections, endocarditis, neonatal sepsis, intra-abdominal and pelvic infections, and endodontic and medical device–associated infections.^[2-6] Of more than 50 species known, Enterococcus faecalis and E. faecium together account for the majority of approximately 90% of clinical isolates (E. faecalis 80–85% and E. faecium 10–15%).^[1-3] Other less commonly isolated species include E. gallinarum, E. casseliflavus, E. avium, E. durans, E. raffinosus, E. mundtii, and E hirae, accounting for approximately 5 to 10% infections.^[1,3,7,8]

Therapy of infections caused by enterococci is problematic because of their intrinsic reduced susceptibility to several frequently used antimicrobial agents such as aminoglycosides (except for high-level resistance), clindamycin, cephalosporins, and trimethoprim/sulfamethoxazole.^[9-11] Moreover, acquired resistance through lateral gene transfer to other agents, (β-lactams, macrolides, glycopeptides, and oxazolidinones) with subsequent emergence of multidrug-resistant (MDR), high-level aminoglycoside-resistant (HLAR) and glycopeptide-resistant enterococci (GRE), including vancomycin-resistant enterococci (VRE), make it more challenging.^[9-11] VRE infections are associated with higher mortality, longer hospital stay, and higher costs compared with vancomycin-susceptible isolates and are recognized as a leading cause of outbreaks of hospital-acquired infections and intensive care unit (ICU) hospitalized patients.^[12-15] Of nine types of vancomycin-related operons/genetic elements (vanA, vanB, van C1/C2/C3, vanD, vanE, vanG, vanL, vanM, and vanN), associated with glycopeptide-resistance in enterococci, van A, and van B are by far the most prevalent types and E. faecium is the predominant species of GRE.^[8-11]

It is important to perform accurate molecular identification of van types along with accurate species identification since at times, enterococci exhibit different phenotypic profile of glycopeptide-resistance which may pose infection control problems.^[16-18] For example, vanA genotype VRE strains exhibiting vanB phenotype pattern have been reported from South Korea, Japan, China, as well as India.^[16-18] Sometimes, unexpected outbreaks with an unanticipated van type may occur representing a change in local epidemiology and necessitating major changes in infection control policies and responses.^[19] A recent study, in fact, has highlighted the importance of adjusting for E. species when assessing the burden of vancomycin resistance.^[20] Additionally, though linezolid and daptomycin have been the drug of choice for management of infections caused by VRE,^[8,10,11,21] both linezolid- and daptomycin-resistant enterococci have emerged recently with simultaneous resistance to both vancomycin and linezolid, as well as to vancomycin and daptomycin.^[8,22-25]

Study of another aspect of enterococcal infections, that is, the pathogenic mechanisms or virulence factors (VFs) is gaining importance as the process of invasion is usually facilitated by damage to host tissues and presence of VFs such as adhesins, colonization factors, and cell aggregates, such as biofilms.^[2,5,26,27] The various VFs encoded by their respective genetic elements consist of both extracellular proteases, as well as cell surface–associated proteins of which gelatinase (gelE), hyaluronidase (hyl), cytolysin (cylA), enterococcal surface protein (esp), accessory colonization factor (ace), aggregation factor (asa1), and endocarditis antigen (efaA) have been studied most intensively.^[26-29] Phenotypic characteristics, such as hemolysis and biofilm formation, have also been recognized as critical for in vivo bacterial growth.^[26-29] Some studies show a relation between the presence of virulence genes and multiple antibiotic resistance, whereas others speculate that virulence genes did not affect the pattern of antimicrobial resistance.^[30-32] Hence, we undertook this study to determine the current pattern of species distribution, antimicrobial susceptibility, and virulence determinants among clinical isolates of enterococci.

Methods

The study, approved by Institutional Ethical Committee, was conducted over a period of 1 year from April 2017 to March 2018 in a tertiary-care research, referral, and teaching hospital in Eastern India.

Isolate Identification and Susceptibility Testing

Consecutive, nonrepeated, discrete, and clinically significant isolates of E. species identified by standard microbiological techniques were included in the study.^[1,3]

Identification was based on the typical magenta-colored colonies on the MacConkey agar, gram-positive reaction, catalase-negativity, growth on and blackening of bile-esculin agar, growth in the presence of 6.5% sodium chloride, heat tolerance test, motility testing, pigment production, and various biochemical tests including arginine dihydrolase reaction and carbohydrate fermentation reactions in purple broth. Susceptibility testing to antimicrobial agents was performed as per the latest Clinical and Laboratory Standards Institute guidelines using discs of standard concentration.^[33] Susceptibility to ampicillin, vancomycin, teicoplanin, linezolid, daptomycin, and fosfomycin was confirmed by gradient minimum inhibitory concentration (MIC) method with EzyMIC strips (HiMedia, Mumbai, India; ►Fig. 1A). HLAR included detection of both high-level gentamicin resistance (HLGR) and high-level streptomycin resistance (HLSR) using discs of gentamicin (120 µg) and streptomycin (300 µg) and confirmed by EzyMIC (gentamicin MIC ≥ 500 μg/mL and streptomycin MIC ≥ 2,000 μg/mL).^[33] Strains with intermediate resistance were included in the percentage of resistant isolates. Multidrug-resistance was defined as nonsusceptibility to at least one agent in three or more antimicrobial categories.^[34] Standard strains of E. faecalis ATCC 29212 (vancomycin susceptible), E. faecium ATCC 35667 (vancomycin susceptible), E. faecalis ATCC 51299 (vancomycin-resistant and HLAR), and E. casseliflavus ATCC 700327 were used as controls.

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Fig. 1

Enterococcus species showing (A) susceptibility to various antimicrobial agents and (B) hemolysin activity.

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Results

Discussion

The present study provides an estimate of the recent pattern of species distribution, antimicrobial susceptibility, and virulence trait profiles of clinical enterococcal isolates in an Indian tertiary care hospital. Frequency of isolation was predominantly from urine specimens followed by wound exudates or blood, as has been observed in other studies from India and abroad.^{[18,27,37,38]}E. faecalis as the overall predominant isolate is congruent with previously published literature.^[37-43] The proportion of E. faecium (28.8%), however, appears moderately high in our institute. This might be due to the increased use of antibiotics expected in a tertiary care institute such as ours which selects out the more resistant species. Recent studies from India and outside have reported rising rates of E. faecium as high as 44.5% (49/110) to 48.3% (80/178).^[39,43] At other places, however, E. faecium still constitutes only approximately 4 to 10% of the enterococal isolates.^[41,42]E. hirae was found as the predominant non-E. faecalis and non-E. faecium isolate in the current study comprising of 4.5% of the total isolates. E. hirae as one of the non-E. faecalis and non-E. faecium isolates from clinical specimens has been described only on few instances before, ranging from 1.6 to 3.03%.^[37,39-41] So the relative distribution of Enterococcus may vary from place to place and also between the institutions. As in previous studies, majority isolates were from admitted patients and ICUs.^[18,44] In Iran, the frequency of VREs isolated from ICUs, nephrology, and internal wards were 33.3, 20.8, and 16.7%, respectively.^[44] In an Indian setting, 291 (79.3%) of 367 isolates were obtained from inpatients with rest from outpatients.^[18]

A high resistance rate to various antimicrobials (erythromycin, ciprofloxacin, levofloxacin, and doxycycline) was observed in the current study which is a cause of concern and precludes their use in routine treatment of enterococcal infections in this region. On the other hand, moderate-to-low resistance was observed to nitrofurantoin (20.2%), fosfomycin (5.2%), and linezolid (4.5%) and none to daptomycin. These latter antimicrobials may therefore be indicated for treatment of enterococcal infections, especially nitrofurantoin and fosfomycin may be recommended for empirical treatment of urinary tract infection due to E. species in our region. Similar high resistance to various antimicrobials has been observed in Iran, Egypt, Turkey, and in another hospital in Eastern India.^{[38,42,43,45]}

Multidrug-resistance, as well as HLAR was observed in 63.1% isolates (HLGR in 53.1% and HLSR in 33.3%) in our study. HLGR and HLSR were detected in 50 and 34% isolates in the study from Iran with MDR observed in 36%.^[42] In Egypt, all E. faecium and 74.6% of E. faecalis were MDR with HLGR detected in 79.6% and HLSR seen in 36.9% isolates.^[38] Frequency of HLAR in India ranges from 47.41 to 72.47%.^[32,39] Since enterococcal resistance to gentamicin and streptomycin occur by different mechanisms of enzymatic inactivation, it is important to test susceptibilities to both agents. Prevalence of GRE (6.5%) is comparable to previous Indian studies which have detected a VRE rate of 7.09 to 8.7%.^[18,32] However, recent studies from Western and North-East India identified higher rates of vancomycin resistance (14.6 and 24%, respectively) with E. faecium accounting for the majority of GRE infections.^[46,47] VRE frequency in other studies outside India ranges from 4.5 to 21%.^[42,43] A point of note is that, similar to our finding, only vanA gene was detected among GRE in various studies from India and outside.^{[27,42,46,48,49]} Interestingly, a recent study from Egypt has described the presence of only vanB and vanC₁ gene clusters in VRE isolates.^[38]

Daptomycin seems to be an alternative therapeutic option for GRE with over 99.8% isolates worldwide being susceptible from 2009 to 2013.^[21,23] None of 47 VRE obtained from rectal, blood, and urine samples from Turkey were resistant to daptomycin.^[49] Recently, however, the proportions of daptomycin-resistant E. faecalis and E. faecium were 3.23 and 10.53%, respectively, in a national collaborative study performed in Spain.^[23] Linezolid, fosfomycin, and chloramphenicol are some of the other few agents that retain in vitro activity against many strains of multiple-drug resistant E. species.^[50,51] Praharaj et al found 37.5% of VRE isolates to be susceptible to chloramphenicol; same has been observed in the current study.^[18] With regard to the linezolid, though it is highly active against gram-positive cocci (GPC) and has good tissue penetration, the rapid emergence of linezolid-resistant GPC is alarming and requires ongoing surveillance. Recent literature review shows linezolid resistance varying from 0.2 to 9.7% among enterococci.^[22,38]

Analysis of the virulence traits in the current study showed that majority of the virulence-encoding genes (efa, ace, asa1, gelE, and cylA) were significantly more prevalent in E. faecalis compared with E. faecium (p < 0.05), with only esp and hyl genes more prevalent in E. faecium. These findings are in accordance with previous reports which state the predominant association of esp and hyl genes with E. faecium and that of the other genes with E. faecalis.^{[28,30,44,52,53]} The esp gene was also significantly more prevalent (p = 0.05) among VRE than among the VSE in Malaysia, with six of seven (85.7%) VRE versus 95 of 215 (44.2%) VSE isolates carrying the gene.^[53] In fact, “esp” is considered as a marker for an epidemic clone of E. faecium that has spread across the countries.^[54] Overall, our results are similar to a study in Turkey, wherein efa gene was the most frequently detected virulence gene (92.7%), followed by ace (83.6%) in 110 isolates and all except hyl were significantly higher in E. faecalis isolates (p < 0.05).^[43] The least prevalent virulence-encoding gene in the current study was hyl which was detected in only seven (6.3%) isolates and may have little role in pathogenicity in comparison to other genes.

As regard to the phenotypic virulence traits, 31.61 and 26.12% of 310 enterocccal isolates in a study from North India demonstrated hemolysis and biofilm production, respectively, slightly lower than in the current study.^[32] In Egypt, the ability to form a biofilm was detected in almost all clinical isolates examined (97/103, 94.2%) with vancomycin- and linezolid-resistant enterococci more likely to exhibit strong/moderate biofilm formation than vancomycin- and linezolid-sensitive ones.^[38] This difference in behavior could be due to local strain-to-strain variation between different geographical regions or different rates of adaptability of the isolates to the local environments. Overall, we found an inverse relationship between antimicrobial resistance and virulence traits; the frequency of majority of virulence traits being lower in isolates displaying higher resistance to antibiotics.

Conclusion

In view of increasing resistance to glycopeptides in enterococci and emerging resistance to currently available alternative therapeutic options for GRE, such as linezolid and fosfomycin, the susceptibility status of various antibiotics among clinical E. species isolates needs to be investigated periodically. To prevent infection and transmission of virulent and resistant enterococcal isolates in the hospital setting, appropriate surveillance and strict infection control measures need to be followed. The present study will contribute to the existing limited data on virulence trait characterization of clinical E. species isolates in India. At the same time, it will help to serve as a guide in the choice of empirical therapy in enterococal infections leading to favorable clinical outcomes by decreasing the clinical failure, microbiological persistence, and associated mortality and will lead to future studies on controlling the spread of virulent and multi-resistant isolates.