Molecular Characterization of High-Level Aminoglycoside Resistance among Enterococcus Species

Introduction

Enterococci have emerged as an important multidrug-resistant nosocomial pathogen causing health-care-associated infections ranging from urinary tract infection, to surgical site infection, prosthetic valve endocarditis, and sepsis. They are highly resilient and versatile, which make them adaptive and survive in the health care environments.^[1]

Two species Enterococcus faecalis and Enterococcus faecium cause the majority of enterococcal infections. They exhibit multidrug resistance by both intrinsic and extrinsic mechanisms. Intrinsically they are resistant to common antibiotics like cephalosporins, penicillinase-resistant penicillin, low-level aminoglycosides, clindamycin, sulfamethoxazole, and trimethoprim. Extrinsically they acquire resistance to high-level aminoglycoside, high-level ampicillin, and vancomycin either through mutations or horizontal transfer of resistant genes.^[1]

Enterococci can develop resistance to aminoglycoside by two different mechanisms, one is the low-level resistance which is due to reduced cell wall permeability and this type can be overcome by using a combination of aminoglycoside and cell-wall-acting agents. Another mechanism is the high-level resistance (HLR) which is due to the production of aminoglycoside-modifying enzymes (AMEs). This enzyme in enterococci negates the synergistic activity of aminoglycoside when it is being combined with a cell-wall-acting agent.^[2,3]

Previously aac(6′)-Ie-aph(2′′)-Ia was the only gene found to be associated with high level gentamicin resistance (HLGR). But in recent years three new AME genes that mediate HLGR in enterococci have been detected, namely aph(2′′)-Ib, aph(2′′)-Ic, and aph(2′′)-Id. Resistance to other aminoglycosides like high level streptomycin and high level kanamycin are usually mediated by aph(3′)-IIIa gene but not to gentamicin. Ant(4′)-Ia gene is also usually associated with high level Aminoglycoside resistance (HLAR).^[2,4-9]

Studies on prevalence of these resistance genes are limited. The purpose of this study is to determine the rate of HLARs and their genetic mechanism in clinical isolates of enterococci. Also, to screen for other common genes that encode for HLAR.

Materials and Methods

Polymerase Chain Reaction

Two sets of multiplex and one simplex polymerase chain reactions (PCRs) were performed using the previously described primers and conditions for all the study isolates. The multiplex primers used for different sets of genes, their annealing temperature, and the amplicon size are listed in ►Table 1.

Table 1 Genes and their sequences for aminoglycoside-modifying enzymes used in PCR

Multiplex set	Gene	Primer sequence (5′ → 3′)	Amplicon size (bp)	Annealing temperature (°C)
1	aac(6′)-Ie-aph(2′′)-Ia	F-CAGGAATTTATCGAAAATGGTAGAAAAG R-CACAATCGACTAAAGAGTACCAATC	369	58
	aph(3′)-IIIa	F-GGCTAAAATGAGAATATCACCGG R-CTTTAAAAAATCATACAGCTCGCG	523	58
2	aph(2′′)-Ib	F-CTTGGACGCTGAGATATATGAGCAC R-GTTTGTAGCAATTCAGAAACACCCTT	867	58
	aph(2′′)-Ic	F-CCACAATGATAATGACTCAGTTCCC R-CCACAGCTTCCGATAGCAAGAG	444	58
	aph(2′′)-Id	F-GTGGTTTTTACAGGAATGCCATC R-CCCTCTTCATACCAATCCATATAACC	641	58
Simplex	ant(4′)-Ia	F-CAAACTGCTAAATCGGTAGAAGCC R-GGAAAGTTGACCAGACATTACGAACT	294	58

Abbreviation: PCR, polymerase chain reaction.

Each reaction volume contained 2 μL of the DNA template added to the master mix which includes 10 pmol of the forward and reverse primers (Sigma-Aldrich, Missouri, United States), 10 Mm dNTPs (Takara, Shiga, Japan), 5 U taq polymerase (Takara, Shiga, Japan), and 10X buffer with MgCl₂ (Takara, Shiga, Japan).

Amplification reactions were performed under the following conditions: initial denaturation at 95°C for 5 minutes, followed by 32 cycles of denaturation at 95°C for 60 seconds, annealing at 58°C for 45 seconds, and elongation at 72°C for 60 seconds, repeated for 32 cycles and a final extension at 72°C for 5 minutes. The PCR product was then run on a 1.5% agarose gel for detection of the amplified fragment (►Fig. 1).

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Fig. 1

Image of gel electrophoresis of PCR for detecting aminoglycoside modifying enzyme (AME) gene. Band at 523bp (T1&T2) represents the presence of aph(3′)-llla gene and band at 369bp (T3) represents presence of aac(6′)-le-aph(2′′)-la gene. L1 is the 100bp ladder. PCR, polymerase chain reaction.

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Results

Out of the 150 isolates, 130 (86.6%) were E. faecalis and 20 (13.3%) were E. faecium. They were obtained from exudates (pus and wound swabs) 95 (63.3%), urine 52 (34.6%), and blood 3 (2%) (►Table 2).

By the Kirby–Bauer disc diffusion method, sensitivity percentages to antibiotics tested are as follows: ampicillin 75% (113/150), high-level gentamicin 48.7% (73/150), erythromycin for isolates from exudates 13% (12/95), vancomycin 98% (147/150), linezolid 100% (150/150), nitrofurantoin 98% (51/52), and ciprofloxacin 46% (24/52).

Minimal inhibitory concentration (MIC) by the agar dilution method revealed high-level gentamicin (> 500 µg/mL) resistance in 51.3% (77/150) isolates (►Fig. 2).

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Fig. 2

Image of minimum inhibitory concentration (MIC) testing for high level gentamicin (> 500 µg/mL); done by the agar dilution method.

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PCR screening for AME genes revealed that 111 of the 150 isolates harbored one or more AME-encoding genes. This distribution is as follows: 11.3% (17/150) isolates harbored aac(6′)-1e-aph(2′′)-1a gene alone, 18.6% (28/150) isolates carried aph(3′)-111a gene alone, and 44.1% (66/150) isolates co-harbored both the above genes. The other AME genes were not detected in this study (►Table 3).

Discussion

Of the 150 isolates collected 130 (86.6%) were E. faecalis and 20 (13.3%) were E. faecium. This reflects the preponderance of E. faecalis over E. faecium among the pathogenic enterococcal species. Almost all the studies had E. faecalis as the predominant isolate except few studies, like a study conducted in Michigan by Vakulenko et al^[4] in which E. faecium was the predominant species. This is in alignment with various other studies conducted across India where E. faecalis has been predominant accounting for 80 to 85%.

Of the 150 isolates, 77 (51.3%) were identified to be resistant to high-level gentamicin phenotypically. Majority of the HLGR enterococci were from urine 47 (61%), followed by exudates 30 (39%). There was no HLGR enterococci isolated from blood stream. The speciation revealed that 60 (78%) were E. faecium and 17 (22%) were E. faecalis. This is in concordance with the previous study from Chennai by Padmasini et al^[2] where E. faecium 39 (51.3%) was found to have higher rates of HLGR than E. faecalis 32 (42%).

The percentage of HLGR in this study was 51.3%, while a few study had lower incidence of HLGR ranging from 27.7 to 49.2%. Others reported higher incidence of 60% to 68%.

Out of the 150 study isolates, 77 (51.3%) were found to be resistant to high-level gentamicin by the agar dilution method. The results of MIC by the agar dilution method and the Kirby–Bauer disc diffusion method were in concordance. No discrepancy was noted, hence the disc diffusion method can be used as a reliable screening test to detect HLGR in enterococci in a clinical laboratory.

Although a spectrum of AME genes are known to be responsible for HLAR status among Enterococcus species, in this study only aac(6′)-1e-aph(2′′)-1a and aph(3′)-llla were encountered among the six genes screened for. Among the study isolates, 55.3% (83/150) isolates had aac(6′)-1e-aph(2′′)-1a gene and 62.6% (94/150) isolates had aph(3′)-llla gene. Varying distribution of both the genes has been cited in the medical literature: aac(6′)-1e-aph(2′′)-1a (38.5–80%); aph(3′)-llla (40–40.4%). The coexistence of these genes was noted in 44% (66/150) isolates in the current study, which is twice that of the previous study from Chennai by Padmasini et al,^[2] which had only 20.2%. Other major AME genes like aph(2′′)-1b, aph(2′′)-1c, aph(2′′)-1d, and ant(4′)-1a were not detected in this study. Previous studies from India had also reported similar AME gene profile with only aac(6′)-1e-aph(2′′)-1a and aph(3′)-llla genes being detected.^[2,10] This observation emphasizes the restricted gene distribution and transfer of resistance gene confined to a geographical region.

Studies from abroad, like the study by Diab et al from Egypt, have observed the presence of aminoglycoside-modifying gene aac (6′)-Ie-aph (2′′)-Ia only in 66.7% of their HLGR isolates and aph (3′)-IIIa gene in 86.5% of high-level streptomycin resistance isolates. aph (2′)-Ib, aph (2′)-Ic, and aph (2′)-Id were not detected.^[11]

Moussa et al characterized HLAR for the presence of AME. The bifunctional AME gene aac(6′)-Ie-aph(2′′)-Ia that confers HLR to gentamicin was detected in 40% of E. faecalis and E. faecium isolates, whereas 32% carried aph(3′)-IIIa. Other AME genes such as aph(2″)-Ib, aph(2″)-Ic, and aph(2″)-Id were not detected in their study as well.^[12]

Elsewhere in Michigan, Vakulenko et al^[4] detected the presence of all majorly prevalent AME genes: aac(6′)-Ie-aph(2′′)-Ia, aph(2′′)-Ib, aph(2′′)-Ic, aph(2′′)-Id, aph(3′)-IIIa, and ant(4′)-Ia. Of the 93 gentamicin-resistant isolates, all contained either the aac(6′)-Ie-aph(2′′)-Ia, aph(2′′)-Ib, aph(2′′)-Ic, or aph(2′′)-Id gene and one isolate carried both aac(6′)-Ie-aph(2′′)-Ia and aph(2′′)-Ic. The aph(3′)-IIIa gene was present in 80 of 113 isolates, and the ant(4′)-Ia gene was present in 26 of 113 isolates. Five of the 20 isolates with low-level resistance to gentamicin contained none of the six genes studied.

In the present study, though 77 (51.3%) of the isolates were phenotypically resistant to gentamicin, 13(8.6%) did not harbor the majorly prevalent AME genes. It may be proposed that they may harbor genes other than those screened for in this study. In contrast, 47 (31.3%) harbored the AME genes but phenotypically appeared susceptible to gentamicin. Possibly they were not expressed, and this observation is in concordance with a previous study from Chennai.^[2]

Conclusion

Enterococcus faecalis is more common than E. faecium among clinical isolates of enterococci. For detection of HLGR, the performance of the disc diffusion susceptibility test is similar to MIC determination by agar dilution. Hence the disc diffusion test can be used as a reliable screening test for HLGR in clinical microbiology laboratory. The most common AMEs mediating HLGR are aac(6′)-1e-aph(2′′)-1a and aph(3′)-111a. This indicates that HLAR genes are widely disseminated among enterococci in our geographical region. This study also emphasizes that the detection of AMEs by PCR is mandatory because strains that appear susceptible by disc diffusion and/or MIC method may harbor one or more AME genes leading to therapeutic failure. And also frequent surveillance studies should be conducted among Enterococcus isolates to document the resistant gene profile.