Background

Enterococci have emerged as an important source of hospital-acquired infections, including those related to the surgical site, respiratory tract, urinary tract, skin and soft tissue infections, and bacteremia. Control and treatment of enterococcal infections are problematic due to their intrinsic resistance to various antimicrobials, their capabilities to develop new resistance and to survive in the external environment for a long time [1, 2].

Enterococci acquire resistance to a wider range of antimicrobial agents particularly, aminoglycosides, glycopeptides, and beta-lactams. This poses a therapeutic challenge to clinicians as they are left with very few treatment options [3, 4]. A common regimen for the treatment of serious enterococcal infections is the synergistic combination of cell wall inhibitors as vancomycin with aminoglycosides [5].

Although enterococci are intrinsically resistant to low levels of aminoglycosides, high-level resistance to aminoglycosides (HLAR) is mediated by acquisition of genes encoding aminoglycoside-modifying enzymes (AME). High-level gentamicin resistance (HLGR) in enterococci is predominantly mediated by aac (6′)-Ie-aph(2′)-Ia gene, which encodes the bifunctional aminoglycoside modifying enzyme AAC (6′)-APH (2′). The action of such enzyme in enterococci eliminates the synergistic activity of gentamicin when combined with a cell wall active agent, such as ampicillin or vancomycin. Other AME genes conferring gentamicin resistance such as aph (2′)-Ib, aph (2′)-Ic, and aph (2″)-Id have been also detected in enterococci. Furthermore, high-level streptomycin and kanamycin resistance in enterococci are mediated by aph (3′)-IIIa [6].

To our knowledge, limited studies on AME genes profile were done in Egypt. The current study was conducted to investigate the rate of HLAR and to determine aminoglycoside resistance-encoding genes profile in enterococcal isolates from different clinical specimens.

Methods

Bacterial isolates and species identification

A prospective study was conducted from November 2016 to March 2018. A total number of 120 non-repetitive enterococcal isolates were collected from different clinical samples from outpatient clinic and hospitalized patients. The clinical specimens were initially cultured on MacConkey agar (HiMedia, India) and Cysteine-Lactose-Electrolyte-Deficient (CLED) media (Bio-Rad, USA). Isolates of enterococci were identified by Gram staining, colony morphology, catalase test, and growth on Bile Esculin agar (Oxoid, England). All isolates were identified to species level using Vitek2 automated system (bioMérieux, France).

Detection of HLAR in enterococcal isolates

Enterococcus species isolates were screened for HLAR by Kirby-Bauer disc diffusion method using streptomycin (300 μg) and gentamicin (120 μg) discs (Bio-Rad, France), and results were interpreted according to the Clinical and Laboratory Standards Institute [7]. Isolates that revealed HLAR by disc-diffusion method were further tested for determining the minimum inhibitory concentrations (MICs) of streptomycin and gentamicin using the E-test (bioMérieux Mercy, France). Overnight bacterial suspensions of test isolates were adjusted to 0.5 MacFarland turbidity and were plated on Muller Hinton agar (MHA) (HIMEDIA) plates and E-test strips (Bio-Merieux France) were applied. The plates were incubated at 35 °C for 15–18 h. Results were interpreted according to The European Committee on Antimicrobial Susceptibility Testing [8].

Bacterial isolates were stored at − 70 °C in the form of glycerol stock until processed for DNA extraction and molecular analysis of AME [9].

Molecular analysis of aminoglycoside modifying genes by PCR assay

Genomic DNA was extracted from Enterococcus strains by cell lysis using a simple boiling technique [10]. The quality and quantity of DNA were assessed with NanoDrop 1000 (Thermo Scientific, USA). PCR assay for AME genes: aac (6′)-Ie-aph (2″)-Ia, aph (2″)-Ib, aph (2″)-Ic, aph (2″)-Id, and aph (3′)-IIIa, was carried out using 4 μL of bacterial DNA extract, 25 pmol of each primer (Table 1), 200 mM of each dNTP (Promega, Inc., USA), 10 mM KCl PCR buffer, 1.5 mM MgCl2, and 1.5 U Taq polymerase (Gotaq Flexi DNA, M8305, Promega, Inc., USA). Amplification was carried out on a Bio-Rad thermal cycler using standard PCR protocol. The cycling conditions were as follows: 5 min of initial denaturation at 95 °C, followed by 35 cycles of denaturation at 95 °C for 1 min, annealing at 58 °C for 1 min and strand extension at 72 °C for 1 min and a final extension for 10 min [11]. A negative control (lacking DNA) was included in each PCR assay. PCR products were analyzed on a 2% agarose gel stained with ethidium bromide. DNA amplicons were visualized using a gel documentation system (cleaver scientific, UK).

Table 1 Primer sequences for amplification of aminoglycoside resistance genes used in PCR assay
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Results

A total of 120 Enterococcus species isolates were revealed from different clinical samples. The predominant species comprised E. faecalis 68 (56.7%), E. faecium 36 (30%), E. gallinarum 12 (10%), and E. casseliflavus 4 (3.3%). Among the studied 120 Enterococcus species isolates, 50 (41.7%) were HLAR. The highest rate of isolation was from urine specimens 72% (36/50), followed by blood 14% (7/50), pus 6% (3/50), wound swabs 4% (2/50), and both of ascetic fluid and sputum revealed only 1 strain (1/50) for each of them representing 2%.

The 50 HLAR isolates included 78% (39/50) HLGR and 74% (37/50) HLSR isolates by disc-diffusion method. HLAR was confirmed in all 50 Enterococcus species isolates by E test and had MIC values of > 512 μg/ml and > 128 μg/ml for gentamycin and streptomycin respectively.

HLAR was predominant in E. faecalis 60% (30/50) followed by E.faecium 30% (15/50), E. gallinarum 8% (4/50), and E. casseliflavus 2% (1/50) by Vitek2 compact system. The species distribution and specimen source of HLAR Enterococcus strains were listed in Table 2.

Table 2 Distribution of HLAR Enterococcus species from various clinical specimens
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Detection of AME encoding genes was performed for the 50 HLAR Enterococcus species isolates and showed that 39 (78%) were HLGR of which 26/39 (66.7%) of the isolates carried the aac (6′)-Ie-aph (2″)-Ia gene (Table 3, Fig. 1), while from the HLSR 37(74%) Enterococcus species strains, 32/37 (86.5%) carried aph (3′)-IIIa gene (Table 3, Fig. 2). Aminoglycoside resistance genes as aph (2″)-Ib, aph (2′)-Ic, and aph (2′)-Id that encode HLGR could not be detected among the studied isolates.

Table 3 Distribution of aminoglycoside modifying enzyme-encoding genes among Enterococcus species with high-level aminoglycoside resistance
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Fig. 1
figure 1

Agarose gel electrophoresis of amplified product AME gene generated by PCR assay. Lane 1: molecular weight marker (ladder 100 bp); lane (2–7): aac (6′)-Ie-aph(2″)-Ia positive (369 bp). Lane 8: negative control

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Fig. 2
figure 2

Agarose gel electrophoresis of amplified product AME gene generated by PCR assay. Lane 1: molecular weight marker (ladder 100 bp); lanes (2 and 3): aph (3′)-IIIa positive (523 bp); lane (4): negative control

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E. faecalis and E. faecium were the predominant Enterococcus species isolates of the present study. They were found to carry the bifunctional enzyme encoding gene aac (6′)-Ie-aph (2″)-Ia in 17/30 (56.7%) and 8/15 (53.3%) strains respectively and aph (3′)-IIIa gene in 18/30 (60%) and 11/15 (73.3%) strains respectively (Table 3).

The coexistence of aac (6′)-Ie-aph (2″)-Ia and aph (3′)-IIIa genes were revealed in E. faecalis, E. faecium, and E .casseliflavus strains. The two E. gallinarum strains were found to carry aph (3′)-IIIa gene only (Table 4).

Table 4 Aminoglycoside modifying enzyme-encoding genes profile among the 50 high-level aminoglycoside resistance Enterococcus species strains
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Discussion

Enterococci have long been considered as one of the most common causes of nosocomial infections. The rise of drug-resistant strains presents a serious problem to control in enterococcal infections. Several resistant Enterococcus species have been reported, including E. faecalis, E. faecium E. avium, E. durans, E. gallinarum, E. casseliflavus, E. raffinosus, E. mundtii, E. malodoratus, and E. hirae [12]. As previously found in Egypt [5, 13], E. faecalis and E. faecium are the predominant strains revealed in all clinical specimens in the present study.

Aminoglycosides are considered efficient in treating serious infections caused by both Gram-positive and Gram-negative organisms. However, the acquisition of extrinsic resistance to high-level aminoglycoside antibiotics in enterococci renders these strains a serious challenge in clinical settings [14].

Distribution of HLAR in enterococcal isolates varied in different reports in the world. The current study results (41.7%) were comparable to those obtained by El-Ghazawy et al. [5] (35.3%) that studied the prevalence of HLAR in 133 enterococcal strains obtained from different clinical samples at Alexandria Main University Hospital, Egypt. However, higher rates of HLAR enterococci (72%) were obtained by Padmasini et al. [11] in India. Another study by El Mahdy et al. [15] at Mansoura University Hospitals in Egypt showed also higher HLAR results (66.3%) in 80 enterococcal isolates recovered from urine samples. The present study revealed a high prevalence of HLSR strains (74%), although the clinical use of streptomycin for infections caused by enterococci has long been restricted due to intrinsic low-level resistance of the organism [11].

Previous studies by Padmasini et al. [11], Bhatt et al. [16], and Niu et al. [17] have reported that HLGR (42.7%, 65%, and 42.7%) was more common than HLSR (29.8%, 45%, and 27.4%) in all species of isolated enterococci respectively. On the contrary, there was nearly no difference between the prevalence rates of HLGR and HLSR among our studied Enterococcus species isolates. Similar finding was found in Turkey by Kurtgoz et al. [18].

HLAR was found to be more common in E. faecalis and E. faecium [19], on the other hand, Abamecha et al. [20] and Bhatt et al. [16] reported that HLAR was a common problem among E. faecium isolates only. A surveillance study that was conducted in 20 European countries had reported 32% and 22% HLGR and 41% and 49% HLSR among HLAR E. faecalis and E. faecium, respectively [19]. In the current study, HLGR is 70% and 100%, while HLSR is 76% and 67% among E. faecalis and E. faecium respectively. This emphasizes susceptibility differences within different enterococcal species.

In accordance with previous studies [5, 11, 17], the aac (6′)-Ie-aph (2′)-Ia gene (66.7%) and aph (3′)-IIIa (86.5%) were identified as the most common AME genes among HLGR and HLSR strains, respectively. Nevertheless, the prevalence of the previous two genes was variable among these former studies. Padmasini et al. [11] stated that the aac (6′)-Ie-aph (2′)-Ia was found in 68.4% of their HLAR enterococcal isolates which is comparable to our results, while 77.4% carried aph (3′)-IIIa gene. Niu et al. [17] and El Ghazawy et al. [5] revealed also higher rates of aac (6′)-Ie-aph (2″)-Ia gene being 89.3% and 95.7%, respectively, which reflects their finding of higher pervasiveness of HLGR in their isolates.

It is noteworthy that the newer aminoglycoside modifying genes aph (2′)-Ib, aph (2′)-Ic, and aph (2′)-Id that encode HLGR were not detected among the studied strains. This was in agreement with Padmasini et al. [11]. In contrast to our results, aph (2′)-Ic gene was detected by El-Ghazawi et al. [5].

Both aac (6′)-Ie-aph (2″)-Ia and aph (3′)-IIIa genes co-existed in isolated E. faecalis, E. faecium, and E. casseliflavus strains, which was in accordance with results obtained by Padmasini et al. [11] and El Mahdy et al. [15]. However, 9 out of the 50 of HLAR enterococcal strains did not carry any of the formerly tested genes. This may be due to the expression of genes other than genes analyzed in this study.

Conclusion

In conclusion, the high detection rate of HLAR among the studied Enterococcus species and the coexistence of HLGR and HLSR strains provide crucial insights into the necessity of HLAR testing as a routine microbiology procedure. The main AME genes among HLGR and HLSR enterococci were (6′)-Ie-aph (2″)-Ia and aph (3′)-IIIa, respectively. The limited AME-encoding genes among the studied HLAR Enterococcus species highlight the restricted gene distribution and transfer of resistant genes within a geographical region. The implementation of an efficient infection control program and regular surveillance of antimicrobial resistance of enterococci is essential in order to establish a rational antibiotic policy for the better management of enterococcal infections.