The prevalence of aminoglycoside-modifying enzyme and virulence genes among enterococci with high-level aminoglycoside resistance in Inner Mongolia, China

Niu, Haiying; Yu, Hui; Hu, Tangping; Tian, Gailin; Zhang, Lixia; Guo, Xiang; Hu, Hai; Wang, Zhanli

doi:10.1016/j.bjm.2016.04.003

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Brazilian Journal of Microbiology

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Medical Microbiology • Braz. J. Microbiol. 47 (3) • Jul-Sep 2016 • https://doi.org/10.1016/j.bjm.2016.04.003 copy

The prevalence of aminoglycoside-modifying enzyme and virulence genes among enterococci with high-level aminoglycoside resistance in Inner Mongolia, China

Authorship SCIMAGO INSTITUTIONS RANKINGS

ABSTRACT

This study highlights the prevalence of aminoglycoside-modifying enzyme genes and virulence determinants among clinical enterococci with high-level aminoglycoside resistance in Inner Mongolia, China. Screening for high-level aminoglycoside resistance against 117 enterococcal clinical isolates was performed using the agar-screening method. Out of the 117 enterococcal isolates, 46 were selected for further detection and determination of the distribution of aminoglycoside-modifying enzyme-encoding genes and virulence determinants using polymerase chain reaction -based methods. Enterococcus faecium and Enterococcus faecalis were identified as the species of greatest clinical importance. The aac(6')-Ie-aph(2")-Ia and ant(6')-Ia genes were found to be the most common aminoglycoside-modifying enzyme genes among high-level gentamicin resistance and high-level streptomycin resistance isolates, respectively. Moreover, gelE was the most common virulence gene among high-level aminoglycoside resistance isolates. Compared to Enterococcus faecium, Enterococcus faecalis harbored multiple virulence determinants. The results further indicated no correlation between aminoglycoside-modifying enzyme gene profiles and the distribution of virulence genes among the enterococcal isolates with high-level gentamicin resistance or high-level streptomycin resistance evaluated in our study.

Keywords:
Enterococcus; High-level aminoglycoside resistance; Aminoglycoside-modifying enzyme gene; Virulence determinant

Gene	Primers (5'–3')	Size (bp)	Annealing temperature (°C)	Reference
ddl _{E. faecium}	F-TTGAGGCAGACCAGATTGACG R-TATGACAGCGACTCCGATTCC	658	54	Macovei and Zurek¹⁹19 Macovei L, Zurek L. Ecology of antibiotic resistance genes: characterization of enterococci from houseflies collected in food settings. Appl Environ Microbiol. 2006;72:4028-4035.
ddl _{E. faecalis}	F-TCAAGTACAGTTAGTCTTTATTAG R-ACGATTCAAAGCTAACTGAATCAGT	941	54	Macovei et al.¹⁹19 Macovei L, Zurek L. Ecology of antibiotic resistance genes: characterization of enterococci from houseflies collected in food settings. Appl Environ Microbiol. 2006;72:4028-4035.
16S rDNA	F-GGATTAGATACCCTGGTAGTCC R-TCGTTGCGGGACTTAACCCAAC	320	54	Macovei et al.¹⁹19 Macovei L, Zurek L. Ecology of antibiotic resistance genes: characterization of enterococci from houseflies collected in food settings. Appl Environ Microbiol. 2006;72:4028-4035.
aac(6')-Ie-aph(2")-Ia	F-CAGGAATTTATCGAAAATGGTAGAAAAG R-CACAATCGACTAAAGAGTACCAATC	369	56	Emaneini et al.²³23 Emaneini M, Bigverdi R, Kalantar D, et al. Distribution of genes encoding tetracycline resistance and aminoglycoside modifying enzymes in Staphylococcus aureus strains isolated from a burn center. Ann Burns Fire Disasters. 2013;26:76-80.
aph(2")-Ib	F-CTTGGACGCTGAGATATATGAGCAC R-GTTTGTAGCAATTCAGAAACACCCTT	867	56	Emaneini et al.²³23 Emaneini M, Bigverdi R, Kalantar D, et al. Distribution of genes encoding tetracycline resistance and aminoglycoside modifying enzymes in Staphylococcus aureus strains isolated from a burn center. Ann Burns Fire Disasters. 2013;26:76-80.
aph(2")-Ic	F-CCACAATGATAATGACTCAGTTCCC R-CCACAGCTTCCGATAGCAAGAG	444	56	Emaneini et al.²³23 Emaneini M, Bigverdi R, Kalantar D, et al. Distribution of genes encoding tetracycline resistance and aminoglycoside modifying enzymes in Staphylococcus aureus strains isolated from a burn center. Ann Burns Fire Disasters. 2013;26:76-80.
aph(2")-Id	F-GTGGTTTTTACAGGAATGCCATC R-CCCTCTTCATACCAATCCATATAACC	641	56	Emaneini et al.²³23 Emaneini M, Bigverdi R, Kalantar D, et al. Distribution of genes encoding tetracycline resistance and aminoglycoside modifying enzymes in Staphylococcus aureus strains isolated from a burn center. Ann Burns Fire Disasters. 2013;26:76-80.
aph(3')-IIIa	F-GGCTAAAATGAGAATATCACCGG R-CTTTAAAAAATCATACAGCTCGCG	523	56	Emaneini et al.²³23 Emaneini M, Bigverdi R, Kalantar D, et al. Distribution of genes encoding tetracycline resistance and aminoglycoside modifying enzymes in Staphylococcus aureus strains isolated from a burn center. Ann Burns Fire Disasters. 2013;26:76-80.
ant(6')-Ia	F-ACTGGCTTAATCAATTTGGG R-GCCTTTCCGCCACCTCACCG	577	56	Sepúlveda et al.²²22 Sepúlveda M, Bello H, Domínguez M, Mella S, Zemelman R, González G. Molecular identification of aminoglycoside-modifying enzymes among strains of Enterococcus spp. isolated in hospitals of the VIII Region of Chile. Rev Med Chil. 2007;135:566-572.
cylA	F-TGGATGATAGTGATAGGAAGT R-TCTACAGTAAATCTTTCGTCA	517	54	Reviriego et al.²¹21 Reviriego C, Eaton T, Martín R, et al. Screening of virulence determinants in Enterococcus faecium strains isolated from breast milk. J Hum Lact. 2005;21:131-137.
gelE	F-AGTTCATGTCTATTTTCTTCAC R-CTTCATTATTTACACGTTTG	402	56	Duprè et al.²⁰20 Duprè I, Zanetti S, Schito AM, Fadda G, Sechi LA. Incidence of virulence determinants in clinical Enterococcus faecium and Enterococcus faecalis isolates collected in Sardinia (Italy). J Med Microbiol. 2003;52(Pt 6):491-498.
esp	F-TTACCAAGATGGTTCTGTAGGCAC R-CCAAGTATACTTAGCATCTTTTGG	936	56	Duprè et al.²⁰20 Duprè I, Zanetti S, Schito AM, Fadda G, Sechi LA. Incidence of virulence determinants in clinical Enterococcus faecium and Enterococcus faecalis isolates collected in Sardinia (Italy). J Med Microbiol. 2003;52(Pt 6):491-498.
ace	F-AAAGTAGAATTAGATCCACAC R-TCTATCACATTCGGTTGCG	320	56	Duprè et al.²⁰20 Duprè I, Zanetti S, Schito AM, Fadda G, Sechi LA. Incidence of virulence determinants in clinical Enterococcus faecium and Enterococcus faecalis isolates collected in Sardinia (Italy). J Med Microbiol. 2003;52(Pt 6):491-498.
agg	F-CCAGTAATCAGTCCAGAAACAACC R-TAGCTTTTTTCATTCTTGTGTTTGTT	406	53	Duprè et al.²⁰20 Duprè I, Zanetti S, Schito AM, Fadda G, Sechi LA. Incidence of virulence determinants in clinical Enterococcus faecium and Enterococcus faecalis isolates collected in Sardinia (Italy). J Med Microbiol. 2003;52(Pt 6):491-498.
efaA	F-CGTGAGAAAGAAATGGAGGA R-CTACTAACACGTCACGAATG	499	56	Duprè et al.²⁰20 Duprè I, Zanetti S, Schito AM, Fadda G, Sechi LA. Incidence of virulence determinants in clinical Enterococcus faecium and Enterococcus faecalis isolates collected in Sardinia (Italy). J Med Microbiol. 2003;52(Pt 6):491-498.

AME gene	Distribution of high-level gentamicin resistance in enterococci (n = 28)			Total no. (%) of isolates
	E. faecium (n = 19)	E. faecalis (n = 7)	E. avium (n = 2)
aac(6')-Ie-aph(2")-Ia	18	5	2	25 (89.3%)
aph(2")-Ib	–	–	–	–
aph(2")-Ic	–	2	–	2 (7.1%)
aph(2")-Id	2	–	1	3 (10.7%)
aph(3')-IIIa	6	1	–	7 (25.0%)
aac(6')-Ie-aph(2")-Ia+aph(2")-Ic	–	2	–	2 (7.1%)
aac(6')-Ie-aph(2")-Ia+aph(2")-Id	2	–	1	3 (10.7%)
aac(6')-Ie-aph(2")-Ia+aph(3')-IIIa	6	–	–	6 (21.4%)

AME gene	Distribution of high-level streptomycin resistance in enterococci (n = 14)		Total no. (%) of isolates
	E. faecium (n = 12)	E. faecalis (n = 2)
ant(6')-Ia	9	1	10 (71.4%)
aph(3')-IIIa	7	1	8 (57.1%)
ant(6')-Ia + aph(3')-IIIa	6	1	7 (50.0%)

Enterococcal species	Strain	AME gene profile
E. faecium	6	aac(6')-Ie-aph(2")-Ia
	13	aac(6')-Ie-aph(2")-Ia+ant(6')-Ia
	15	aac(6')-Ie-aph(2")-Ia+aph(2")-Id
	20	aac(6')-Ie-aph(2")-Ia+ant(6')-Ia
	21	aac(6')-Ie-aph(2")-Ia+aph(3')-IIIa+ant(6')-Ia
	29	aac(6')-Ie-aph(2")-Ia+ant(6')-Ia
	30	aac(6')-Ie-aph(2")-Ia+aph(3')-IIIa+ant(6')-Ia
	34	aac(6')-Ie-aph(2")-Ia+aph(3')-IIIa+ant(6')-Ia
	43	aac(6')-Ie-aph(2")-Ia+aph(3')-IIIa
	49	aac(6')-Ie-aph(2")-Ia+aph(3')-IIIa+ant(6')-Ia
	51	aac(6')-Ie-aph(2")-Ia+aph(2")-Id+aph(3')-IIIa+ant(6')-Ia

E. faecalis	44	aac(6')-Ie-aph(2")-Ia+aph(2")-Ic+aph(3')-IIIa+ant(6')-Ia

Virulence gene	Distribution of high-level aminoglycoside resistance in enterococci (n = 30)			Total no. (%) of isolates
	E. faecium (n = 20)	E. faecalis (n = 8)	E. avium (n = 2)
gelE	4	6	0	10 (33.3%)
esp	2	4	1	7 (23.3%)
cylA	3	4	0	7 (23.3%)
ace	0	1	0	1 (3.3%)
agg	0	0	0	0 (0%)
efaA	0	8	0	8 (26.7%)

Sociedade Brasileira de Microbiologia USP - ICB III - Dep. de Microbiologia, Sociedade Brasileira de Microbiologia, Av. Prof. Lineu Prestes, 2415, Cidade Universitária, 05508-900 São Paulo, SP - Brasil, Ramal USP 7979, Tel. / Fax: (55 11) 3813-9647 ou 3037-7095 - São Paulo - SP - Brazil
E-mail: bjm@sbmicrobiologia.org.br

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[1] *Corresponding authors. E-mails: btyxyhh@163.com (H. Hu), wang.zhanli@hotmail.com (Z. Wang).