High Prevalence of the aac(6′)-Ie-aph(2′′)-Ia Gene in Hospital Isolates of Enterococcus faecalis Co-Resistant to Gentamicin and Penicillin

Abstract

Objectives:

This study aimed to characterize the molecular mechanism of resistance to gentamicin among penicillin-resistant, ampicillin-susceptible Enterococcus faecalis (PRASEF) isolates by investigating the presence of the aac(6′)-Ie-aph(2′′)-Ia gene. The co-resistance to antimicrobials of other classes was also evaluated.

Results:

Among the 151 isolates evaluated, 70 were PRASEF and 81 were penicillin-susceptible and ampicillin-susceptible E. faecalis (PSASEF). No β-lactamase producing isolate was detected. Eighty-three (55.0%) and 35 (23.2%) out of the 151 E. faecalis isolates showed high-level gentamicin resistance (HLGR) and high-level streptomycin resistance (HLSR) phenotypes. However, a significantly higher rate of PRASEF (88.6%) showed HLGR phenotype in comparison with PSASEF (23.5%) (p < 0.01). Conversely, a significantly lower rate of PRASEF (14.3%) showing HLSR was observed in comparison with PSASEF (30.9%) (p = 0.02). The prevalence of isolates displaying multidrug resistance (MDR) phenotype was significantly higher (p < 0.01) in the group of PRASEF (81.4%) than in PSASEF (18.6%). The majority of PSASEF (61.9%) and PRASEF (90.3%) isolates showing HLGR phenotype was harboring the aac(6′)-Ie-aph(2′′)-Ia gene, which encodes a bifunctional enzyme that inactivates all aminoglycosides except streptomycin.

Conclusion:

The aac(6′)-Ie-aph(2′′)-Ia gene was prevalent among the Brazilian PRASEF isolates that usually exhibit co-resistance to gentamicin and to multiple other drugs.

Introduction

In recent decades, enterococci have emerged as an effective opportunistic pathogen causing a variety of infections worldwide, especially in hospitals.^1,2 In Europe, enterococci are currently the third most frequently isolated bacteria from health care-associated infections.³ These microorganisms are intrinsically resistant to several antimicrobial agents and have a great ability to acquire and express new resistance determinants besides producing multiple virulence traits, which facilitate their persistence in hospital environment and survival to many host defenses.⁴ Of the enterococcal species clinically relevant, Enterococcus faecalis is the most predominant causing 80–90% of infections.⁵

All enterococci exhibit intrinsic resistance to low levels of aminoglycosides due to inefficient active transport of aminoglycosides across the cytoplasmic membrane, but these drugs are still clinically useful in combination with compounds that act on the bacterial cell wall, such as beta-lactams or vancomycin, generating a synergistic bactericidal effect.² However, the emergence of strains with acquired resistance to one of these antimicrobials compromises the effectiveness of the bactericidal synergism.^6,7

The first description of high-level aminoglycoside resistance (HLAR) in E. faecalis was reported in 1979 in France and since then has spread worldwide.⁸ In the 1990s, high-level gentamicin resistance (HLGR) was detected in various European countries, with the prevalence varying from 1% to 48%.⁹ Currently, the overall percentage of HLGR in E. faecalis in Europe is 30.5%, with national percentages varying between 12.5% and 56.3%.¹⁰

Aminoglycoside resistance occurs through different mechanisms but the most clinically relevant is due to enzymatic inactivation of the antimicrobial molecule by the action of aminoglycoside modifying enzymes (AMEs) called aminoglycoside acetyltransferases (AACs) and adenylyltransferases or nucleotidyltransferases (ANTs) and phosphotransferases (APHs), which are carried on mobile genetic elements (MGEs) that are widespread among enterococci.^11,12

A remarkable variety of types and subtypes of AMEs have been reported, although the bifunctional aac(6′)-Ie-aph(2′′)-Ia enzyme is the most widely distributed.^7,11,12 This enzyme is coded by the aac(6′)-Ie-aph(2′′)-Ia gene and confers resistance to gentamicin (high-level) and to virtually all of the clinically available aminoglycosides, except for streptomycin. Therefore, testing for HLAR in enterococci has required only the use of gentamicin and streptomycin. The types and distribution of AMEs and their genes in enterococci vary according to geographic regions and are still poorly studied in Brazil.

Resistance to penicillins and other beta-lactam antibiotics is rare in E. faecalis isolates. However, in 2006, it was reported the emergence of penicillin-resistant, ampicillin-susceptible E. faecalis (PRASEF) clinical isolates in a hospital in Greece.¹³ Thereafter, E. faecalis isolates with this unusual phenotype of resistance to beta-lactams have also been described in other countries, including Brazil.^14–17 Interestingly, it has been reported that PRASEF isolates tend to show co-resistance to gentamicin, the most commonly used aminoglycoside against enterococci.^13–15 Therefore, the aim of this study was to characterize the molecular mechanism of gentamicin resistance among PRASEF isolates by investigating the presence of the aac(6′)-Ie-aph(2′′)-Ia gene. The co-resistance to antimicrobials of other classes was also evaluated.

Materials and Methods

Bacterial isolates and patients

One hundred and fifty one nonrepetitive isolates of E. faecalis recovered from patients admitted to Hospital de Clínicas of the Federal University of Triângulo Mineiro, located in Uberaba, Minas Gerais, Brazil, in the period of February 2006 to June 2016, were evaluated in the present study. Of those isolates, 70 were PRASEF and 81 were penicillin- and ampicillin susceptible (PSASEF). A few isolates (34 PRASEF and 15 PSASEF) included in this study belonged to previous publications.^15,18 The species identification of isolates was based on phenotypic tests.⁵ The species were also confirmed by PCR using specific primers as described elsewhere.¹⁹ All the isolates were preserved at −70°C in brain heart infusion broth containing 20% glycerol.

The isolates were recovered from various clinical samples mainly urine (57; 37.7%), wounds (44; 29.1%), and normally sterile body fluids (blood, pleural fluid, cerebrospinal fluid, and peritoneal fluid) (34; 22.5%). The age of patients ranged from newborn to 97 years, but the great majority aged above 50 years (58.9%). Fifty-eight patients (38.0%) evaluated were females and 92 (62.0%) were males. Patients were hospitalized mainly on the following hospital wards: surgical (44; 29.1%), medical (33; 21.8%), and intensive care (31; 20.5%).

This study was approved by the Research Ethics Committee of the Federal University of Triângulo Mineiro (document number 1365).

Antimicrobial susceptibility testing

The antimicrobial minimal inhibitory concentrations (MICs) for the beta-lactams (penicillin and ampicillin) and the aminoglycosides (gentamicin and streptomycin) were determined by E-test (AB bioMérieux, Solna, Sweden) and by the broth dilution method, which is considered the reference method. Mueller-Hinton broth (Difco, Becton, Dickinson and Company Sparks, France) and solutions of antimicrobials prepared from powders of known potencies (Sigma–Aldrich Denmark A/S, Copenhagen, Denmark) were used in broth dilution method. The susceptibility testing was performed and interpreted according to the Clinical and Laboratory Standards Institute guidelines.²⁰ For penicillin and ampicillin, MIC ≥16 μg/mL indicated resistance. For gentamicin and streptomycin, MIC ≥500 μg/mL and MIC ≥1,000 μg/mL, respectively, indicated resistance.

The antimicrobial susceptibility of E. faecalis isolates was also tested using the disk diffusion method. The disks (Oxoid, England) of antimicrobials from various classes were used: penicillin (10 μg) and ampicillin (10 μg) (β-lactams); gentamicin (120 μg) and streptomycin (300 μg) (aminoglycosides); ciprofloxacin (5 μg) (fluoroquinolone); vancomycin (30 μg) (glycopeptide), erythromycin (15 μg) (macrolide); tetracycline (30 μg) (tetracycline), and chloramphenicol (30 μg) (phenicol).

Disk diffusion and E-test results for the beta-lactams and aminoglycosides were compared with those obtained by broth dilution method and interpretative discrepancy as very major errors (VME) (false susceptible) and major errors (ME) (false resistance) were assessed.

Quality-control testing was performed for all susceptibility tests, using E. faecalis ATCC^® 29212 and E. faecalis ATCC 51299, according to CLSI guideline recommendation.²⁰ The isolates were defined as multidrug resistant if they exhibited acquired resistance to at least one representative agent in three or more different classes of antimicrobial in addition to gentamicin or streptomycin.

β-Lactamase production

β-lactamase production was tested with the chromogenic nitrocefin disk (Becton, Dickinson and Company, Cefinase™), and the Staphylococcus aureus ATCC 25923 was used as positive control.

Amplification of the aac(6′)-Ie-aph(2′′)–Ia gene

Bacterial DNA from the E. faecalis isolates and the reference strains (E. faecalis ATCC 29212 and ATCC 51299) was extracted using the QIAamp^® DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's recommendations. The aac(6′)-Ie-aph(2′′)-Ia gene was amplified by PCR using two pairs of specific primers that generated amplicons of 369 and 348 bp as described previously,²¹ with minor modifications. The PCR amplification products were separated by 1.5% agarose gel electrophoresis and visualized after ethidium bromide staining under UV light.

Statistical analysis

Data were analyzed using the SPSS statistical software package (version 17.0; SPSS, Inc., Chicago, IL). The chi-squared test was used to compare categorical data. All tests were two tailed, and a p-value ≤0.05 was considered statistically significant.

Results

In Table 1, the E-test and broth dilution MIC values obtained for the beta-lactams and aminoglycosides tested according to the groups of isolates evaluated are summarized, PRASEF (n = 70) and PSASEF (n = 81), which were determined based on the results of the reference method (broth dilution). No β-lactamase producing isolate was detected.

Table 1.

Beta-lactams and Aminoglycoside MIC Range, MIC50, and MIC90 Values Obtained by Different Susceptibility Methods for the Groups of Penicillin-Resistant (n = 70) and Penicillin-Susceptible (n = 81) Enterococcus faecalis Isolates

Antimicrobial	Method	E. faecalis group	MIC (μg/mL) range	MIC50 (μg/mL)	MIC90 (μg/mL)
Penicillin	BD	Pen-S	2–8	4	8
	BD	Pen-R	16–32	16	32
	E-test	Pen-S	1.5–8	2	8
	E-test	Pen-R	12–64	16	32
Ampicillin	BD	Pen-S	0.5–4	1	2
	BD	Pen-R	0.5–8	4	8
	E-test	Pen-S	0.25–4	0.75	2.0
	E-test	Pen-R	1–8	4	6
Gentamicin	BD	Pen-S	≤62.5–>2,000	≤62.5	>2,000
	BD	Pen-R	≤62.5–>2,000	>2,000	>2,000
	E-test	Pen-S	12–>1,024	16	>1,024
	E-test	Pen-R	8–>1,024	>1,024	>1,024
Streptomycin	BD	Pen-S	≤125–>4,000	≤125	>4,000
	BD	Pen-R	≤125–>4,000	≤125	1,000
	E-test	Pen-S	64–>1,024	256	>1,024
	E-test	Pen-R	96–>1,024	128	1,024

MIC, minimal inhibitory concentration; BD, broth dilution; Pen-S, penicillin-susceptible; Pen-R, penicillin-resistant.

The penicillin MIC values by broth dilution of PRASEF isolates were not high, ranging 16–32 μg/mL. Ampicillin MICs against PRASEF isolates were below the CLSI susceptible breakpoints, but they were significantly higher than those for PSASEF isolates in both susceptibility methods (E-test and broth dilution) (p < 0.01). No significant difference was observed regarding the MIC results obtained with E-test for either penicillin or ampicillin in both groups of PRASEF and PSASEF isolates. VME (n = 3) and ME (n = 4) were observed using the disk diffusion method for penicillin, while only ME (n = 7) were observed for ampicillin. The MIC results obtained with E-test were also concordant with those obtained with broth dilution method for both aminoglycosides tested (Table 1), but VME (n = 8 and n = 3) and ME (n = 5 and n = 6) were observed using disk diffusion method for gentamicin and streptomycin, respectively.

Among the 151 E. faecalis isolates studied, 83 (55.0%) and 35 (23.2%) showed HLGR and high-level streptomycin resistance (HLSR) phenotypes, respectively. Twenty-one (13.9%) isolates showed resistance to both, gentamicin and streptomycin, simultaneously. However, as demonstrated in Table 2, a significantly higher rate of PRASEF isolates (88.6%) showed co-resistance to gentamicin in comparison with those PSASEF (25.9%) (p < 0.01). Conversely, a significantly lower rate of PRASEF isolates (14.3%) showing co-resistance to streptomycin was observed in comparison with PSASEF isolates (30.9%) (p = 0.02).

Table 2.

Antimicrobial Susceptibility Profile of the Groups of Penicillin-Resistant and Penicillin-Susceptible Enterococcus faecalis Isolates

Antimicrobial	Number (%) of resistant isolates
Antimicrobial	Pen-S (n = 81)	Pen-R (n = 70)	Total (n = 151)
Chloramphenicol	22 (27.2)	60 (85.7)	82 (54.3)
Ciprofloxacin	53 (65.4)	67 (95.7)	120 (79.5)
Erythromycin	61 (75.3)	69 (98.6)	130 (86.1)
Gentamicin	21 (25.9)	62 (88.6)	83 (55.0)
Streptomycin	25 (30.9)	10 (14.3)	35 (23.2)
Tetracycline	41 (50.6)	63 (90.0)	104 (68.9)

p-Values were <0.01 for all antimicrobials tested except for streptomycin (p = 0.02) comparing the resistance rates between the groups of penicillin-resistant and penicillin-susceptible Enterococcus faecalis isolates.

Overall, the majority of E. faecalis isolates showed resistance to antimicrobials of other classes such as erythromycin (86.1%), ciprofloxacin (79.5%), tetracycline (68.9%), and chloramphenicol (54.3%), whereas all of them were susceptible to vancomycin. Note that significantly (p < 0.01) higher resistance rates were observed for all antimicrobials in the group of PRASEF except for streptomycin that shows a significantly (p = 0.02) lower resistance rate compared to that of the PSASEF group (Table 2). The antimicrobial co-resistance patterns are shown in Table 3 according to the group of isolates evaluated (PRASEF and PSASEF). The prevalence of isolates displaying MDR phenotype was significantly higher (p < 0.01) in the group of PRASEF (n = 60; 85.7%) than in PSASEF (n = 26; 32.1%).

Table 3.

Antimicrobial Resistance Patterns According to the Groups of Penicillin-Resistant and Penicillin-Susceptible Enterococcus faecalis Isolates

Multidrug resistance	Antimicrobial resistance	Number (%) of resistant isolates
Multidrug resistance	Antimicrobial resistance	Pen-S (n = 81)	Pen-R (n = 70)
Non-MDR	None	7 (8.6)
	CHL	1 (1.2)
	CIP	6 (7.4)
	ERY	4 (4.9)
	CIP, ERY	6 (7.4)
	CIP, TET	2 (2.4)
	ERY, TET	3 (3.7)
	STR, CIP	1 (1.2)
	STR, ERY	1 (1.2)
	STR, GEN	1 (1.2)
	CHL, CIP, ERY	2 (2.4)
	CHL, CIP, GEN	1 (1.2)	1 (1.4)
	CHL, ERY, GEN		1 (1.4)
	CHL, ERY, TET	2 (2.4)	1 (1.4)
	CIP, ERY, GEN		1 (1.4)
	CIP, ERY, STR	2 (2.4)
	CIP, ERY, TET	5 (6.1)	2 (2.8)
	ERY, GEN, STR	1 (1.2)
	ERY, GEN, TET	1 (1.2)
	ERY, STR, TET	4 (4.9)
	CHL, CIP, ERY, TET	5 (6.1)	4 (5.7)
Total		55 (67.9)	10 (14.3)
MDR	CIP, ERY, GEN, STR	2 (2.4)	1 (1.4)
	ERY, GEN, STR, TET	2 (2.4)	1 (1.4)
	CIP, ERY, GEN, TET	2 (2.4)	2 (2.8)
	CIP, ERY, STR, TET	2 (2.4)	1 (1.4)
	CHL, CIP, ERY, GEN	1 (1.2)	2 (2.8)
	CHL, ERY, STR, TET	1 (1.2)
	CHL, CIP, ERY, GEN, TET	5 (6.1)	46 (65.7)
	CHL, CIP, ERY, STR, TET	5 (6.1)
	CIP, ERY, GEN, STR, TET	4 (4.9)	2 (2.8)
	CHL, CIP, ERY, GEN, STR		1 (1.4)
	CHL, CIP, ERY, GEN, STR, TET	2 (2.4)	4 (5.7)
Total		26 (32.1)	60 (85.7)

All isolates were susceptible to vancomycin and ampicillin.

MDR, multidrug resistance; CHL, chloramphenicol; CIP, ciprofloxacin; ERY, erythromycin; GEN, gentamicin; STR, streptomycin; TET, tetracycline.

As demonstrated in Table 4, the majority of PSASEF (61.9%) and PRASEF (90.3%) isolates showing the HLGR phenotype were harboring the aac(6′)-Ie-aph(2′′)-Ia gene (p<0.01). However, this gene was not detected in some HLGR isolates, especially in those of the PSASEF group (38.1%) in comparison with the PRASEF group (9.7%) (p = 0.01). Moreover, 11.7% and 12.5% of PSASEF and PRASEF isolates, respectively, which exhibited the non-HLGR phenotype, was carrying the aac(6′)-Ie-aph(2′′)-Ia gene (p = 0.67).

Table 4.

Distribution of the aac(6′)-Ie-aph(2′′)-Ia Gene Among the Enterococcus faecalis Isolates with High-Level Gentamicin Resistance Phenotype and According to the Penicillin Susceptibility Profile

E. faecalis group	Gentamicin phenotype	Number (%) of isolates	aac(6′)-Ie-aph(2′′)-Ia		p-value
E. faecalis group	Gentamicin phenotype	Number (%) of isolates	Present	Absent	p-value
Pen-S (n = 81)	non-HLGR	60 (74.1)	7 (11.7)	53 (88.3)	<0.01
Pen-S (n = 81)	HLGR	21 (25.9)	13 (61.9)	8 (38.1)
Pen-R (n = 70)	non-HLGR	8 (11.4)	1 (12.5)	7 (87.5)	<0.01
Pen-R (n = 70)	HLGR	62 (88.6)	56 (90.3)	6 (9.7)

Discussion

Acquired resistance to aminoglycosides (high level), penicillins, and glycopeptides is the most clinically important in enterococci because the synergistic combination of these compounds results in the killing of the organism that is essential for the treatment of those more severe infections. In this study, we highlight that PRASEF isolates that were reported recently usually exhibit co-resistance to gentamicin (high level), which was demonstrated to be associated with the presence of the aac(6′)-Ie-aph(2′′)-Ia gene. Moreover, these E. faecalis isolates resistant to penicillin were typically MDR showing co-resistance to multiple antimicrobials in addition to gentamicin, reducing the therapeutics options for treatment of infections.

It is known that PRASEF isolates usually present reduced susceptibility to other beta-lactam drugs such as imipenem and piperacillin in addition to penicillin.^13–15,17 Moreover, although these isolates are still susceptible to ampicillin according to the actual susceptible breakpoints the ampicillin MIC values against PRASEF isolates are higher than those ones against penicillin-susceptible isolates as demonstrated in this and in previous studies.^15,17 The mechanisms that lead to this incongruous phenotype of resistance to penicillin but susceptible to ampicillin in E. faecalis were not fully comprehensive to date although point mutation in pbp4 (protein-binding penicillin) gene has been reported in PRASEF isolates.^17,18 The PBP4 acts in the final stages of peptidoglycan synthesis and has low affinity for β-lactam antibiotics similar to PBP5 found in E. faecium.⁶

In this study, PRASEF isolates were typically co-resistant to multiple classes of antimicrobials although they were still susceptible to vancomycin. Note that co-resistance is the outcome of the accumulation of resistance determinants to different classes of antimicrobials achieved by chromosomal mutations and/or acquisition of MGEs, which are very common in enterococci.²² Overall, E. faecalis isolates of hospital origin are commonly resistant to the macrolides, tetracyclines, fluoroquinolones, and chloramphenicol.^6,23 However, it is noteworthy that PRASEF isolates showed even higher rates of resistance to these drugs than the PSASEF isolates, although the latter have also been recovered in the same hospital.

Interestingly, while the gentamicin co-resistance rate was significantly higher among PRASEF (88.6%) in comparison with PSASEF (25.9%) isolates similar to the other drugs tested, the streptomycin co-resistance rate was significantly lower (14.3 and 30.9, respectively). This fact could be related to the molecular mechanisms of resistance to these aminoglycosides and/or the location of the resistance determinants in PRASEF genomes. In some previous studies, HLGR was also found to be more prevalent than HLSR phenotype in E. faecalis and in other enterococcal species as well.^24,25

Varied prevalence rates of HLGR in E. faecalis have been reported in different geographic regions or countries although recent surveillance studies evaluating high number of isolates are scarce. In Andean America, the HLGR rates reported in E. faecalis were 69% in Peru, 44% in Colombia, 32% in Ecuador, and 13% in Venezuela.²⁶ HLGR was reported in 44.0% of E. faecalis isolates from Italy,²⁷ in 42.2% from Iran,²⁸ and in 66.1% from China²⁹; lower HLGR rates have been reported from Japan (34.0%)²⁵ and Australia (34.8%).³⁰ In this study, the overall HLGR phenotype rate was 55.5% similar to that reported previously in some countries and in Brazil.³¹ However, when the E. faecalis was stratified according to the penicillin resistance profile, a much higher rate of HLGR (88.6%) was observed among the PRASEF isolates. Note that such high HLGR rate in E. faecalis has not been reported elsewhere to date.

Mostly, E. faecalis isolates showing HLGR phenotype (PRASEF and PSASEF) in this study carried the bifunctional AME gene aac(6′)-Ie-aph(2′′)-Ia, which is in accordance with Padmasini et al.³² and Zhang et al.²⁹ who reported 68.4% and 97.3% presence of this gene, respectively. Several other studies conducted in different countries also indicated that aac(6′)-Ie-aph(2′′)-Ia gene is the most prevalent gene among the gentamicin-resistant enterococci.^7,11,12 Unexpectedly, we detected few E. faecalis isolates that did not show HLGR phenotype despite harboring aac(6′)-Ie-aph(2′′)-Ia similar to those reported in previous studies conducted in China, India, and Japan.^29,32,33 Note that in all these studies the phenotypic method used for testing gentamicin resistance was the broth dilution, considered the golden standard, similar to the present study.

The reasons for the nonexpression of this gene are unknown, and the clinical significance of the non-HLGR isolates harboring aac(6′)-Ie-aph(2′′)-Ia has not been investigated. Therefore, one may speculate that there is a risk of onset of HLGR during the treatment considering that Watanabe et al.³³ demonstrated that both sequences of the gene and its promoter region were identical among E. faecalis isolates showing HLGR and non-HLGR phenotype. Moreover, some HLGR isolates were not harboring aac(6 ′)-Ie-aph (2 ”)-Ia gene in this study as previously reported.²⁹ This phenomenon may be due to the action of other less frequent AMEs that were not investigated in this study or to the different resistance mechanisms. In contrast, one may also consider the intrinsic limitations of any PCR assay.

The global dissemination of the aac(6′)-Ie-aph(2′′)-Ia gene among E. faecalis has been explained by its location on MGEs such as conjugative transposons (Tn) that mediate the horizontal transfer of resistance determinants and through the population of enterococci species. The Tn5281 has been frequently demonstrated to carry the aac(6′)-Ie-aph(2′′)-Ia gene in HLGR E. faecalis isolates.^29,33–35 Other genes conferring resistance to different drugs were also found on MGEs.³⁵ Recently, it has been reported that pbp5, which is responsible for ampicillin and penicillin resistance in E. faecium similar to pbp4 in E. faecalis, is located on a large chromosome platform that is horizontally transferable and has other metabolic and adaptive characteristics.³⁶

Taking into account all these considerations we speculate that the co-resistance to gentamicin and penicillin in E. faecalis isolates may be due to the presence of these MGEs, which probably have been accumulated in PRASEF isolates. Furthermore, it is noteworthy that PRASEF isolates from Brazil and from Denmark, which were also co-resistant to gentamicin, belong to the clonal complexes (CCs) denominated CC9 and CC6, as determined by multilocus sequence typing.^14,18 Both CCs, CC9 and CC6 (formerly CC2), are characterized by encompassing hospital-adapted lineages of E. faecalis that are globally dispersed.^18,37 Differently, it was demonstrated that HLGR E. faecalis isolates harboring the aac(6′)-Ie-aph(2′′)-Ia from Japan belonged to different CCs not included in the four major global CCs (CC6, CC9, CC10, and CC21), except for one isolate from CC2.^33,37

In conclusion, the findings of this study demonstrate that the aminoglycoside resistance aac(6′)-Ie-aph(2′′)-Ia gene is prevalent among the Brazilian PRASEF isolates that usually exhibit co-resistance to gentamicin (high level) and to multiple other drugs. Further studies are warranted to evaluate which MGEs are involved in the co-resistance of penicillin with gentamicin in these MDR E. faecalis isolates, which represent a high risk of accumulating resistance to other antimicrobials.

Footnotes

Disclosure Statement

The authors declare that they have no conflicts of interest.

Funding Information

This work was supported by Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) - (Grant CBB - APQ-01672-13). F.E.L.C. was supported by a doctoral fellowship from CAPES.

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