| Abstract|| |
Background: Several enterococcal species are increasingly being reported from clinical infections, besides the major species. Aim: This study was undertaken to determine the prevalence of unusual enterococcal species and their antimicrobial susceptibility patterns, virulence factors, and molecular characterization. Study Design and Settings: The study was conducted in Department of Microbiology and associated Tertiary Care University Hospital in North India. Materials and Methods: Enterococcal isolates were collected for a period of 2 years from clinical specimens. Identification and elaborate phenotypic characterization was done biochemically. All the isolates were tested by Kirby-Bauer disc diffusion method and breakpoint minimum inhibitory concentration for susceptibility against standard antibiotics. Screening for vancomycin-resistant enterococci (VRE), high-level aminoglycoside resistance was done on brain heart infusion agar incorporated with 6 μg/ml vancomycin, 500 μg/ml gentamicin, and 2000 μg/ml streptomycin, respectively. VRE isolates were tested for the presence of vanA, vanB, and vanC genes and high-level gentamicin resistant (HLGR) isolates for aac-6'- aph-2' gene by polymerase chain reaction (PCR). Hemolysin and gelatinase production, hemagglutination and biofilm formation were detected along with asa1, gelE, esp, hyl, and cylA genes by multiplex PCR. Results: Of 403 enterococci, 93 (23.07%) isolates were identified as unusual species and atypical variants. Resistance of 52.68%, 46.23%, 44.08%, and 6.45% for ampicillin, ciprofloxacin, high strength gentamicin, and vancomycin, respectively were noted. Presence of vanC gene in Enterococcus gallinarum and Enterococcus casseliflavus isolates and vanA gene in Enterococcus durans and Enterococcus hirae and aac-6'- aph-2'' gene was found in 33.14% (14/41) of the HLGR isolates. The most frequent virulence factor was biofilm production. Only a few isolates harbored asa1 (2), gelE (9), and hyl (3) genes. Conclusion: Considerable prevalence of pathogenic unusual species of enterococci was seen along with their emerging drug resistance and virulence. Complete identification and routine speciation is essential to limit their emergence as major species in near future.
Keywords: Antimicrobial, emergence, enterococci, species, unusual
|How to cite this article:|
Tuhina B, Anupurba S, Karuna T. Emergence of antimicrobial resistance and virulence factors among the unusual species of enterococci, from North India. Indian J Pathol Microbiol 2016;59:50-5
|How to cite this URL:|
Tuhina B, Anupurba S, Karuna T. Emergence of antimicrobial resistance and virulence factors among the unusual species of enterococci, from North India. Indian J Pathol Microbiol [serial online] 2016 [cited 2019 Jun 25];59:50-5. Available from: http://www.ijpmonline.org/text.asp?2016/59/1/50/174795
| Introduction|| |
Enterococci have evolved over the years to become an important etiogen in nosocomial and community-acquired infections. Along with the emergence of antimicrobial resistance and their widespread dissemination, there has been a simultaneous emergence of several species of enterococci, increasingly being isolated from clinical samples. Twenty-three species of Enterococcus have been associated with clinical significance, with Enterococcus faecalis and Enterococcus faecium being the two major species.  However, due to several issues like intrinsic resistance to certain group of antibiotics and serving as potential reservoirs of transferable resistant elements, the non-faecalis and non-faecium isolates are increasingly being recognized as a cause of worldwide concern. For this reason, identification and characterization of enterococcal isolates from clinical samples has become important.
Other than antimicrobial resistance, the emergence of several virulence traits to adapt to the host defense mechanisms has occurred in enterococci, primarily in the major species. However, it has also been seen that these virulence traits have gradually penetrated the genetic lineages of other enterococcal species,  thus assisting in their dissemination. Knowledge of these virulence traits is essential to control their early emergence.
Most of the studies have dealt with the two major species, not taking into account the exact prevalence of the other species. Importance of the unusual species was realized in cases of serious infections like endocarditis or when their nosocomial transmission became evident, thus requiring epidemiological surveillance within the hospital environment.  With this background, the following study was undertaken to determine the prevalence of unusual enterococcal species, isolated from clinical samples along with their antimicrobial susceptibility patterns, virulence factors, and molecular characterization.
| Materials and methods|| |
Isolation and identification of study isolates
Enterococcal isolates were collected for a period of 2 years, from September 2010 to August 2012, from patients attending the outpatient and inpatient services of the various departments of a 1200 bedded Tertiary Care University Hospital in North India. The study was approved by the Institute Ethical Committee. Clinical samples namely urine, blood, body fluids, wound swabs, and exudates, were plated on cystine lactose electrolyte deficient agar or MacConkey agar and blood agar as per the nature of the specimen. Presumptive identification of enterococci was made based on Gram staining, bile esculin hydrolysis, growth at pH 9.6, pyrrolidonyl (PYR) hydrolysis test , and growth in 6.5% sodium chloride. 
Further, the elaborate phenotypic characterization was done based on conventional biochemical tests described by Facklam and Collins.  Carbohydrate fermentation tests were used in 1% broth base. Mannitol, sorbitol, sorbose, arabinose, raffinose, lactose, sucrose (Sigma, USA), and pyruvate (Hi Media, India) fermentation was tested. Deamination of arginine was detected in Moeller's decarboxylase media (Hi Media, India), while motility was seen in sulphide-indole-motility media and confirmed by hanging drop preparation. Pigment formation was seen on trypticase soy agar (Difco, USA). Further, to differentiate atypical nonlactose fermenting variants of E. faecalis from Enterococcus solitarius, isolates were grown on blood tellurite agar. 
Antimicrobial susceptibility testing
All the clinical isolates were tested by Kirby-Bauer disc diffusion method using ampicillin (10 μg), ciprofloxacin (5 μg), nitrofurantoin (300 μg, for urinary isolates only), high strength gentamicin (HSG, 120 μg), vancomycin (30 μg), and linezolid
(30 μg) (Hi Media, India) discs.  Screening for vancomycin-resistant enterococci (VRE), high-level aminoglycoside resistance was done on brain heart infusion (BHI) agar incorporated with 6 μg/ml vancomycin, 500 μg/ml gentamicin, and 2000 μg/ml streptomycin, respectively, based on standard protocol.  Specific breakpoint minimum inhibitory concentration (MIC) by agar dilution method for ampicillin (resistant MIC ≥16 μg/ml) and ciprofloxacin (resistant MIC ≥4 μg/ml) resistance was determined. MIC for vancomycin and teicoplanin was performed for the VRE isolates by agar dilution method.  E. faecalis ATCC 29212 was used as a control.
Molecular characterization of drug resistant isolates
The vancomycin-resistant isolates as detected by screen agar were further tested for the presence of vanA, vanB, and vanC resistant determinants by polymerase chain reaction (PCR) as described elsewhere.  The high-level gentamicin resistant (HLGR) isolates were tested for the presence of the bifunctional enzyme aac-6'- aph-2' gene. 
Detection of virulence factors
Phenotypic detection of virulence factors namely hemolysin production on 5% human blood agar,  gelatinase production on Todd-Hewitt agar containing 3% gelatin,  hemagglutination activity  and biofilm formation by semiquantitative assay  were performed. Briefly, isolates showing beta hemolysis on 5% human blood agar following 24 h incubation were considered as hemolysin producers. Gelatinase production was interpreted as a zone of clearing around the colonies on Todd-Hewitt agar containing 3% gelatin, after addition of Frazier's solution. Hemagglutination activity was seen by mixing isolates from BHI broth to 3% washed human erythrocyte in microwells for agglutination. Biofilm formation was seen in sterile polystyrene microtitre plates (Tarsons, India). Enterococcal isolates were grown in BHI broth containing 2% glucose in these wells. After washing and staining, optical density of each well was measured at 450 nm in an automated ELISA reader and interpreted for semiquantitative biofilm formation.
Genotypic detection of virulence factors namely asa1, gelE, esp, hyl, and cylA genes encoding for the respective virulence factors aggregation substance, gelatinase, cytolysin, enterococcal surface protein, and hyaluronidase was performed by multiplex PCR as given elsewhere.  All the primers used in this study has been tabulated in [Table 1].
| Results|| |
A total of 403 isolates of enterococci were collected from various clinical samples over a period of 2 years, of which majority belonged to E. faecalis and E. faecium (310, 76.92%). Remaining 93 isolates were identified as unusual species and atypical variants of Enterococcus comprising 30 isolates of Enterococcus durans (32.25%), 25 Enterococcus hirae (26.88%), 7 E. solitarius (7.52%), 5 Enterococcus dispar (5.37%), 2 Enterococcus pseudoavium (2.15%), 2 Enterococcus gallinarum (2.15%), 1 Enterococcus casseliflavus (1.07%), 10 isolates of asaccharolytic variants (mannitol negative) E. faecalis (10.75%), and 11 isolates of nonlactose fermenting E. faecalis (11.82%). The non-lactose fermenting variants were differentiated from the biochemically similar E. solitarius by the typical black colonies on blood tellurite medium. The various sources of these isolates have been shown in [Table 2]. The majority of the isolates (85, 91.39%) were from patients with urinary tract infections (UTI).
|Table 2: Distribution of species of enterococci among the clinical isolates |
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Antimicrobial susceptibility testing of these isolates by breakpoint MIC performed by standard agar dilution and screen agar methods revealed varying susceptibilities against various antibiotics as shown in [Table 3]. While ampicillin resistance was >50% in most of the isolates, ciprofloxacin resistance was comparatively lower (46.23%), followed by HSG resistance (44.08%). HLGR (44.08%) was found to be present more commonly than high-level streptomycin resistance (30.1%) in majority of the isolates. The urinary isolates showed good susceptibility to nitrofurantoin (80%). However, majority of the isolates of E. dispar, E. pseudoavium, and E. gallinarum along with the atypical variants were resistant to most of the antibiotics tested. All the isolates were susceptible to linezolid by disc diffusion method.
|Table 3: Resistance pattern of unusual enterococcal isolates from clinical samples by breakpoint MIC method |
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On vancomycin screen agar, 6 isolates were screened to be resistant. The phenotypes of these isolates as interpreted by their MIC values against vancomycin and teicoplanin were in full concordance with their genotype. MIC of vancomycin and teicoplanin were as follows: E. hirae and E. durans vancomycin MIC ≥128 μg/ml, teicoplanin MIC ≥16 μg/ml, E. gallinarum and E. casseliflavus vancomycin MIC ≥8 μg/ml, and teicoplanin MIC ≥0.5 μg/ml.
Vancomycin resistance determinants revealed the presence of vanC gene in all the E. gallinarum and E. casseliflavus isolates. VanA gene was found in a single isolate each of E. durans and E. hirae [Figure 1]. The presence of aac-6'-aph-2''gene was found in 33.14% (14 of 41) of the HLGR isolates with 25%
|Figure 1: Polymerase chain reaction amplification of van genes in the vancomycin resistant enterococci isolates|
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(3 of 12) of E. durans, 22.2% (2 of 9) of E. hirae, 50% (1 of 2) of E. pseudoavium, 33.3% (1 of 3) of E. solitarius, 100% (2 of 2) of mannitol negative E. faecalis, and 50% (5 of 10) of nonlactose fermenting E. faecalis harboring the gene.
Presence of several virulence factors was detected phenotypically. The most frequent factor was biofilm production followed by hemolysin production and hemagglutination as shown in [Table 4]. None of the isolate harbored the cylA and esp gene, whereas all the gelatinase producers carried gelE gene. Among the virulence genes, only a few isolates harbored asa1, gelE and hyl genes [Figure 2].
|Table 4: Phenotypic and genotypic characterization of virulence factors in unusual spp. |
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| Discussion|| |
The emergence of unusual species, as well as a change in the occurrence of the common species, has been recently reported in case of enterococci. It is a well-known fact that for long the ratio of infections of E. faecalis against those due to all other enterococcal species was 10:1. However, a progressive decrease in this ratio has been noted lately especially in cases of enterococcal bacteremia. The predominance of E. faecium species out limiting the previous abundance of E. faecalis has been the major reason responsible for this "microbiologic shift".  Other than these two major species, most often 10 species namely E. avium, E. casseliflavus, E. durans, E. faecalis, E. gallinarum, E. hirae, E. malodoratus, Enterococcus mundtii, E. pseudoavium, Enterococcus raffinosus, and E. solitarius, along with Enterococcus cecorum, Enterococcus columbae, Enterococcus saccharolyticus, E. dispar, Enterococcus sulfureus, Enterococcus seriolicida, and Enterococcus flavescens are attributed to those causing disease. 
This study revealed the emergence of unusual species of enterococci in North India. Initially, a prevalence of 2-10% was attributed to non-faecalis and non-faecium strains.  With the passage of time, detailed phenotypic characterization raised the prevalence to 14.8%  and 19%  , which might reflect the actual scenario. Meanwhile, in a span of few more years, the prevalence in north India as depicted in our study stepped up to 23.07%. Other than a number of case reports about the pathogenic potential of these uncommon species, , their exact prevalence, characterization and importance have rarely been studied. Recently, several molecular methods for detection of these unusual species have been suggested.  However, conventional methods based on physiological and biochemical properties are still most commonly employed for identification and characterization of species as in this study. Majority of the unusual isolates were isolated from urine samples and none from the blood. This might suggest less severe infections caused by these isolates in our setting. Epidemiologically, enterococci rank second to Escherichia coli as uropathogens causing UTI.  Otherwise too, UTI is the most frequent infection caused by enterococci. Therefore, it was expected that most of the isolates were of urinary origin.
Antimicrobial susceptibility testing revealed that 52.68% of these unusual species were resistant to ampicillin, with E. pseudoavium isolates and E. dispar showing 100% and 80% resistance respectively. Beta-lactam agents are usually the first choice of treatment for treating for mild enterococcal infections and in synergy with aminoglycosides for severe infections. However, HLGR was also considerable (44.08%) in these isolates exhibiting 90.9%, 50%, and 60% resistance, respectively. In such situation, a combination therapy could not be given. Ciprofloxacin resistance was 46.23% in the overall isolates. As the majority of them were isolated from urine, ciprofloxacin (or any other fluoroquinolone) still remains a good therapeutic option for UTI by these unusual species. It should be mentioned here that isolates of E. dispar and E. gallinarum along with atypical E. faecalis variants were more resistant to ciprofloxacin in which case other alternatives for treatment should be sought for. Nitrofurantoin remains the only exception to this trend of antimicrobial resistance with only 20% resistance, a fact also shown in other studies. 
Enterococci have marked ability to acquire and inherit drug resistance determinants. Most of these resistant determinants are transposon mediated and are freely transferred from one another.  Emergence of vancomycin resistance was also revealed in these unusual species. A single isolate each of E. durans and E. hirae was found to vancomycin-resistant harboring the vanA gene. While vanA and vanB resistant determinants have been described primarily in E. faecium and E. faecalis, their easy dissemination via transposons is responsible for their free transfer among fellow members. The vanC gene was found in all the isolates of E. gallinarum and E. casseliflavus. vanC gene confers low-level resistance to vancomycin and is intrinsically found in these motile enterococcal species.  Usually, vanC is not transferrable. However, the recent reports of transfer of vanA gene to these intrinsically resistant isolates with the vanC gene are a real cause of concern.  vanA being the most widely distributed gene finds a potent reservoir in these unusual species. Though not, in this case, these intrinsically resistant motile species have a predilection for infection in immunocompromised, and transplant recipients and consequently their identification is of importance.  HLGR in enterococci is mediated by production of aminoglycoside modifying enzymes, of which the most common is the bifunctional enzyme encoded by aac-6'- aph-2''gene.  Enterococcal isolates with this gene is resistant to all aminoglycosides except streptomycin. In this study, this gene was found to have disseminated to varying extent among the unusual species, especially in the atypical variants of E. faecalis. This gene has been found on plasmids and also as part of transposon Tn5281 and is highly transferrable.  However, its absence in the HLGR isolates implies the availability of other aminoglycosides except gentamicin as a therapeutic option.
The study revealed the gradual dissemination of several virulence factors in these unusual pathogens. Gelatinase production, biofilm formation, and hemagglutination activity have often been associated with increased ability to adhere to underlying tissues.  As majority was urinary isolates, expression of these virulence traits can lead to persistence of infections. Of the two major species, it has been seen that E. faecalis possesses more virulence factors than E. faecium. Interestingly, the atypical variants of E. faecalis also expressed several virulence factors.
Though hemolysin production, hemagglutination activity and biofilm formation was exhibited by nearly one-fifth of the isolates, virulent genes were sparsely distributed among them. While esp, which is considered as a marker for an epidemic clone of E. faecium that has spread across the countries,  and cylA, were absent in these isolates, frequency of other virulence genes was low. Similar to drug resistance, genetic lineages with increased virulence due to the acquisition of new virulence traits occur by gene transfer.  Antibiotic resistance determinants, cytolysin toxin production, gelatinase production, aggregation substance, enterococcal surface protein are some of the traits that have infiltrated into the species to varying extent, thus increasing the pathogenicity of enterococci. As majority of the virulent genes are plasmid-borne, their infrequent possession among the unusual enterococcal species probably indicates that the emerging species have not yet faced the widespread dissemination of virulent determinants like the major species. Another explanation of this aspect could be the cost of fitness of these emerging organisms. While the emerging drug resistance in these isolates is sufficient enough for their better survival thus eliminating the requirement of additional virulence traits.
This study showed a considerable prevalence of unusual species of enterococci as pathogens isolated from clinical samples, along with their emerging drug resistance and virulence. Complete identification and routine speciation of these isolates is still a matter of debate, despite increasing evidence of their rising prevalence. Factors like selective antibiotic pressure along with indiscriminate antibiotic use in animal feed have often been implicated for the emergence of these species.  In addition, the high mobility of resistance and virulence determinants among the members of Enterococcus also play a major role in their rapid dissemination. Therefore, on one hand while revealing the wide diversity of enterococcal species in clinical infections is necessary, all measures to control their rapid spread and acquisition of multidrug resistance should be employed to limit the emergence of these isolates as major species in near future.
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Conflicts of interest
There are no conflicts of interest.
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Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi - 221 005, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]