High Level Aminoglycoside Resistance and Distribution of Aminoglycoside Resistant Genes among Clinical Isolates of Enterococcus Species in Chennai, India

Enterococci are nosocomial pathogen with multiple-drug resistance by intrinsic and extrinsic mechanisms. Aminoglycosides along with cell wall inhibitors are given clinically for treating enterococcal infections. 178 enterococcal isolates were analyzed in this study. E. faecalis is identified to be the predominant Enterococcus species, along with E. faecium, E. avium, E. hirae, E. durans, E. dispar and E. gallinarum. High level aminoglycoside resistance (HLAR) by MIC for gentamicin (GM), streptomycin (SM) and both (GM + SM) antibiotics was found to be 42.7%, 29.8%, and 21.9%, respectively. Detection of aminoglycoside modifying enzyme encoding genes (AME) in enterococci was identified by multiplex PCR for aac(6′)-Ie-aph(2′′)-Ia; aph(2′′)-Ib; aph(2′′)-Ic; aph(2′′)-Id and aph(3′)-IIIa genes. 38.2% isolates carried aac(6′)-Ie-aph(2′′)-Ia gene and 40.4% isolates carried aph(3′)-IIIa gene. aph(2′′)-Ib; aph(2′′)-Ic; aph(2′′)-Id were not detected among our study isolates. aac(6′)-Ie-aph(2′′)-Ia and aph(3′)-IIIa genes were also observed in HLAR E. durans, E. avium, E. hirae, and E. gallinarum isolates. This indicates that high level aminoglycoside resistance genes are widely disseminated among isolates of enterococci from Chennai.


Introduction
Enterococci have emerged as an important multiple-drug resistant nosocomial pathogen reported worldwide. Its resistance to wider range of antimicrobial agents particularly, aminoglycosides, glycopeptides and beta-lactams had increasingly been documented [1]. Although enterococci are intrinsically resistant to low levels of aminoglycosides, high level resistance to aminoglycosides (MIC ≥ 2000 g/mL) is mediated by acquisition of genes encoding AMEs. High level gentamicin resistance (MIC ≥ 500 g/mL) in enterococci is predominantly mediated by aac(6 )-Ie-aph(2 )-Ia, which encodes the bifunctional aminoglycoside modifying enzyme AAC(6 )-APH (2 ). The action of this enzyme in enterococci eliminates the synergistic activity of gentamicin when combined with a cell wall active agent, such as ampicillin or vancomycin. Recently, newer AME genes such as aph(2 )-Ib, aph(2 )-Ic, and aph(2 )-Id have been detected as those conferring gentamicin resistance in enterococci. High level streptomycin and kanamycin resistance in enterococci are mediated by aph(3 )-IIIa gene encoding aminoglycoside phosphotransferase enzyme, APH(3 )-IIIa [2]. In India, high level aminoglycoside resistance has been reported from different centers; however, studies on prevalence of these resistance genes are limited. The goal of this study is to determine, the rate of high level aminoglycoside resistance and aminoglycoside resistance encoding genes in enterococcal isolates collected from different specimen sources in Chennai, India.

Bacterial Strains.
A total of 178 nonidentical clinical isolates of enterococci were obtained from clinical specimens from various tertiary care centers from Chennai, during a period of 2010-2012. Appropriate inpatient details were collected and recorded to avoid identical isolates from the 2 The Scientific World Journal

Minimum Inhibitory Concentration for Aminoglycosides.
The isolates were confirmed as high level aminoglycoside resistant enterococci (HLARE) by considering growth ≥512 g/mL for gentamicin and ≥2048 g/mL for streptomycin. The overnight bacterial cultures were adjusted to 0.5 McFarland's turbidity and the inoculum was spot inoculated on the surface of brain heart infusion agar with increasing concentrations of gentamicin and streptomycin antibiotics (HiMedia, Mumbai, India). The plates were incubated at 37 ∘ C for 24 hrs and inspected for more than one colony forming units in the spotted area. Enterococcus faecalis ATCC 29212 was used as a negative control strain.
Amplification was performed with PCR system (Eppendorf, Germany) and the cycling programs consisted of an initial denaturation (95 ∘ C, 5 min) followed by 32 cycles each of denaturation (95 ∘ C, 1 min), annealing (58 ∘ C, 1 min) and extension (72 ∘ C, 1 min), with a final extension of (72 ∘ C, 5 min). Each amplification product was resolved by electrophoresis with a 100-base pair molecular weight marker (Real Biotech Corporation, Taiwan) in a 1.2% agarose-Trisborate-EDTA gel stained with ethidium bromide (0.5 g/mL) and visualized under gel documentation system (BioRad, USA). Table 1 shows the product size of all the genes analyzed.

Identification of Enterococcus Species.
Since early 1970s, Enterococci were considered as nosocomial pathogens. The incidences of high level GM/SM resistance have been disseminated in many Enterococcus species. Since then, the high level aminoglycoside resistance has become a serious problem in most of the health care settings; identification of clinical isolates of enterococci up to species level is essential for an appropriate management of the infection. The predominant species observed in our study was E. faecalis 86/178 (48.3%) as observed in previous studies [6] in our region. Other than E. faecium which was 80/178 (44.9%), we have also obtained E. avium (2%), E. hirae (1.6%), E. durans (0.6%), E. gallinarum, and E. dispar (1%). The species distribution and specimen source of isolates were listed in Table 2.

High Level Aminoglycoside Resistance in Enterococcal
Isolates. Aminoglycoside antibiotics are considered efficient in treating serious infections caused by both gram positive and gram negative organisms. Due to acquisition of extrinsic resistance to high level aminoglycoside antibiotics in enterococci, these strains gain importance in clinical settings. A total of 178 enterococcal isolates were screened by MIC method, 76/178 (42.7%) were HLGR (MIC ≥ 512 g/mL) for gentamicin and 53/178 (29.8%) were HLSR (MIC ≥ 2048 g/mL) for streptomycin (Table 3). Although the clinical use of streptomycin for enterococci has long been restricted due to intrinsic low level resistance (LLR), the present study revealed HLSR strains.
reported 32% and 22% HLGR and 41% and 49% HLSR among gentamicin resistant E. faecalis and E. faecium, respectively [9]. A very recent study conducted in Iran [10] had reported around 60.45% HLGR strains in their region. This is higher than our present report. They were suggesting cotransfer of these resistance genes along with VRE for the higher percentage of HLGR in their study. However, studies on AME gene profile were not done frequently in our region.

PCR Identification of HLAR Genes in Enterococci.
All 178 enterococcal isolates were analyzed for the presence of aminoglycoside modifying enzyme coding genes (Figure 1). High level gentamicin resistance is primarily due to the presence of bifunctional enzyme aac(6 )-Ie-aph(2 )-Ia which also confers high level resistance to amikacin, tobramycin, kanamycin, netilmicin, and dibekacin except streptomycin [11]. aph(2 )-Ib was first detected from E. faecium and E. coli and confers high level resistance to gentamicin, tobramycin, amikacin, kanamycin, netilmicin, and dibekacin but not to amikacin. aph(2 )-Ic confers HLR to gentamicin, tobramycin, and kanamycin while the strains carrying them can be treated with amikacin, netilmicin, and streptomycin in combination with cell wall inhibitors. Earlier this gene was shown to be present in E. gallinarum; but it had also been reported in isolates obtained from farm animals and in E. faecalis and E. faecium [12]. aph(2 )-Id was reported in E. casseliflavus and has similar mechanism to that of aph(2 )-Ib [13]. aac(6 )-Ie-aph(2 )-Ia gene was found in 38.2% of enterococcal isolates in our study. But, out of 76 strains of HLGR identified by MIC method, only 52 strains (68.4%) carried aac(6 )-Ie-aph(2 )-Ia gene. However, 24/76 (31.57%) isolates that were high level gentamicin resistant and 12/53 (22.64%) isolates that were high level streptomycin resistant did not carry any of the genes tested. In a previous study [14], all the high level gentamicin resistant E. faecalis and E. faecium isolates were found to carry aac(6 )-Ie-aph(2 )-Ia gene.
Another most important gene tested in our study, aph (3 )   Each of the HLAR E. avium, E. hirae, and E. durans had both the genes while another E. avium strain carried aph(3 )-IIIa with streptomycin MIC of 1024 g/mL. One of the two E. gallinarum strains isolated was HLAR and it carried both aminoglycoside resistance genes aac(6 )-Ie-aph(2 )-Ia and aph(3 )-IIIa, while the other strain carried aph(3 )-IIIa gene alone but with an MIC of ≥16 g/mL for gentamicin and >64 g/mL for streptomycin. One of the two E. dispar isolates in our study was found to be HLGR + HLSR and carried aph(3 )-IIIa gene (see Table 4).

Conclusion
In our study, we had observed enterococcal isolates with phenotypic resistance towards high level gentamicin and streptomycin antibiotics without presence of respective AME gene. This may be due to the expression of genes other than genes analyzed in this study. The coexistence of aac(6 )-Ieaph(2 )-Ia and aph(3 )-IIIa was observed in 20.2% of the isolates.
Though an array of AMEs are responsible for HLAR status among Enterococcus species, we have demonstrated aac(6 )-Ie-aph(2 )-Ia and aph(3 )-IIIa genes more frequently occurring than other genes. This observation emphasizes the restricted gene distribution and transfer of resistant genes within a geographical region. Hence, surveillance studies should be conducted among Enterococcus isolates from different sources in any given geographical area to document the AME gene profile. Our study is the first to report resistance gene analysis among the Enterococcus species in India.