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Detection of aminoglycoside resistance genes in Riemerella anatipestifer isolated from ducks
Veterinary Microbiology 158 (2012) 451–452
Contents lists available at SciVerse ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Letter to the Editor Detection of aminoglycoside resistance genes in Riemerella anatipestifer isolated from ducks Dear Editor, Riemerella anatipestifer (RA) is the causative agent of a contagious septicaemic disease especially in ducklings, turkeys and other birds. The disease accounts for serious economic losses to the duck industry due to high morbidity and mortality, weight loss and carcase condemnations. Currently, various antibiotics are used to prevent and control RA infection in ducks, but this accelerates the emergence of drug-resistant strains. The resistance of RA to many antibiotics has increased greatly, and some antibiotic resistance genes in RA have been detected (Chang et al., 2003; Zhong et al., 2009; Chen et al., 2010, 2011; Yu et al., 2008; Zheng et al., 2011). Aminoglycoside antibiotics play an important role in the therapy of infections caused by Gram-positive and Gram-negative bacteria. The mechanisms of bacterial resistance to aminoglycosides have been the subject of numerous genetic and biochemical studies (Vakulenko and Mobashery, 2003; Wright, 2005). At present, there are three general mechanisms of aminoglycoside resistance, and enzymatic modification is the most important mechanism (Shaw et al., 1993; Hotta et al., 1996; Hayashi et al., 1997), resulting in a loss of antibacterial activity due to a diminished affinity for the ribosomal A-site target (Llano-Sotelo et al., 2002). These enzymes are assigned to three classes: aminoglycoside nucleotidyltransferases (ant), aminoglycoside acetyltransferases (aac), and aminoglycoside phosphotransferases (aph).These enzymes, including a large number of aminoglycoside-modifying enzymes, are most relevant to clinical resistance in Grampositive organisms (McKay et al., 1996). In this study, the aminoglycoside resistance genes (ant(300 )-Ia, aac(60 )-Ib and aph(30 )-IIa gene) were detected in 38 RA strains isolated from sick ducks in China during April 2005 to October 2011. According to the sequences of aminoglycoside antibiotics resistant genes in E. coli, the following primers were used for the detection of ant(300 )-Ia gene (F-primer, 50 -ATC TGGCTATCTTGCTGACA-30 and R-primer, 50 -TATGACGGGCTGATACTGG-30 ), aac(60 )-Ib gene (F-primer, 50 -ATGACCTTGCGATGCTCTATGA-30 and R-primer, 50 -CGAATGCCTGGCGTGTTT-30 ), and aph(30 )-IIa gene (F-primer, 50 -TGACTGGGCACAACAGACA-30 and R-primer, 50 -TCAAGAAGGCGATAGAAGGC-30 ) (Sun et al., 2011). The amplification program 0378-1135/$ – see front matter ß 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2012.02.027
consisted of an initial step of 10 min at 94 8C, followed by 32 cycles of 50 s at 94 8C, 45 s at 54 8C and 50 s at 72 8C, and a final extension of 10 min at 72 8C. The PCR amplification products were analyzed by 0.8% agarose gel electrophoresis and stained with ethidium bromide. All the positive PCR products were cloned and sequenced, and the nucleotide sequences analyzed by the Clustal W method with DNAstar megalign software (DNAStar Inc., Madison, WI, USA). The aminoglycoside resistance genes were found in all the 38 RA strains, with 36 (95.43%) strains carrying two or three aminoglycoside resistance genes and 2 strains carrying only one aminoglycoside resistance gene (Table 1). A total of 12 (31.58%), 34 (89.47%) and 36 (97.37%) isolates were positive for ant(300 )-Ia, aac(60 )-Ib and aph(30 )-IIa gene, respectively. The nucleotide identity within RA strains was 95.8-100, 97.9-100 and 98.7-100% in ant(300 )-Ia, aac(60 )-Ib and aph(30 )-IIa, respectively. A similar result was also obtained from nucleotide identity between RA and E. coli or Salmonella strains, which was 94.4–100, 98.4–100 and 98.9– 100% in the three genes, respectively. RA, E. coli and Salmonella are common bacterial pathogens in intensive duck husbandry. It is interesting that the three bacteria possess highly related aminoglycoside resistance genes (94.4–100% nucleotide identity). Concluding, ant(300 )-Ia, aac(60 )-Ib and aph(30 )-IIa genes encoding aminoglycoside-modifying enzymes were highly prevalent in RA strains isolated from China, and the three genes had high nucleotide identities with those in E. coIi and Salmonella. These findings highlight the need for further comprehensive study to better understand the mechanisms of aminoglycoside resistance in RA. These newly identified sequences were deposited into the GenBank, with accession numbers JQ664600–JQ664611 Table 1 The detection result of aminoglycoside resistance genes in 38 RA strains. Aminoglycoside resistance genes Negative One resistance gene Two resistance genes Three resistance genes
ant(300 )-Ia aac(60 )-Ib aph(30 )-IIa ant(300 )-Ia + aac(60 )-Ib ant(300 )-Ia + aph(30 )-IIa aac(60 )-Ib + aph(30 )-IIa ant(300 )-Ia + aac(60 )-Ib + aph(30 )-IIa
No. of positive strains
Relevance ratio (%)
0 0 1 1 1 3 24 8
0 0 2.63 2.63 2.63 7.89 63.16 21.05
452
Letter to the Editor / Veterinary Microbiology 158 (2012) 451–452 grons in clinical strains of Riemerella anatipestifer. Vet. Microbiol., doi:10.1016/j.vetmic.2011.11.002.
of ant(300 )-Ia gene, JQ664612–JQ664645 of aac(60 )-Ib gene and JQ664646–JQ664681 of aph(30 )-IIa gene. References
Fang-Fang Yanga,b,1 Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Shandong Agricultural University, Shandong Taian, China b Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong Taian, China a
Chang, C.F., Lin, W.H., Yeh, T.M., Chiang, T.S., Chang, Y.F., 2003. Antimicrobial susceptibility of Riemerella anatipestifer isolated from ducks and the efficacy of ceftiofur treatment. J. Vet. Diagn. Invest. 15, 26–29. Chen, Y.P., Tsao, M.Y., Lee, S.H., Chou, C.H., Tsai, H.J., 2010. Prevalence and molecular characterization of chloramphenicol resistance in Riemerella anatipestifer isolated from ducks and geese in Taiwan. Avian Pathol. 39, 333–338. Chen, Y.P., Lee, S.H., Chou, C.H., Tsai, H.J., 2011. Detection of florfenicol resistance genes in Riemerella anatipestifer isolated from ducks and geese. Vet. Microbiol. 154, 325–331. Hayashi, S.F., Norcia, L.J., Seibel, S.B., Silvia, A.M., 1997. Structure–activity relationships of hygromycin A and its analogs: proteinsynthesis inhibition activity in a cell free system. J. Antibiot. 50, 514–521. Hotta, K., Zhu, C.B., Ogata, T., Sunada, A., Ishikawa, J., Mizuno, S., Kondo, S., 1996. Enzymatic 2-N-acetylation of arbekacin and antibiotic activity of its product. J. Antibiot. 49, 458–464. Llano-Sotelo, B., Azucena Jr., E.F., Kotra, L.P., Mobashery, S., Chow, C.S., 2002. Aminoglycosides modified by resistance enzymes display diminished binding to the bacterial ribosomal aminoacyl-tRNA site. Chem. Biol. 9, 455–463. McKay, G.A., Roestamadji, J., Mobashery, S., Wright, G.D., 1996. Recognition of aminoglycoside antibiotics by the enterococcal–staphylococcal aminoglycoside 30 -phosphotransferase type IIIa: role of substrate amino groups. Antimicrob. Agents Chemother. 40, 2648–2650. Shaw, K.J., Rather, P.N., Hare, R.S., Miller, G.H., 1993. Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes. Microbiol. Rev. 57, 138–163. Sun, H., Lei, Z., Zou, J.F., Wei, Z., Wang, X., Xie, Z.J., Jiang, S.J., 2011. Mechanism of resistance and detection of resistance genes to aminoglycoside among avian Escherichia coli strains from Shandong Province. Chin. J. Vet. Sci. 31, 1279–1282. Vakulenko, S.B., Mobashery, S., 2003. Versatility of aminoglycosides and prospects for their future. Clin. Microbiol. Rev. 16, 430–450. Wright, G.D., 2005. Bacterial resistance to antibiotics: enzymatic degradation and modification. Adv. Drug Deliv. Rev. 57, 1451–1470. Yu, C.Y., Liu, Y.W., Chou, S.J., Chao, M.R., Weng, B.C., Tsay, J.G., Chiu, C.H., Ching Wu, C., Long Lin, T., Chang, C.C., Chu, C., 2008. Genomic diversity and molecular differentiation of Riemerella anatipestifer associated with eight outbreaks in five farms. Avian Pathol. 37, 273–279. Zhong, C.Y., Cheng, A.C., Wang, M.S., Zhu de, K., Luo, Q.H., Zhong, C.D., Li, L., Duan, Z., 2009. Antibiotic susceptibility of Riemerella anatipestifer field isolates. Avian Dis. 53, 601–607. Zheng, F., Lin, G., Zhou, J., Cao, X., Gong, X., Wang, G., Qiu, C., 2011. Discovery and characterization of gene cassettes-containing inte-
Ya-Ni Sun1 College of Veterinary Medicine, Northwest A & F University, Shanxi Yangling, China Jing-Xin Lia,b Hui Wanga,b Meng-Jiao Zhaoa,b Jing Sua,b Zhen-Jun Zhanga,b Hong-Jie Liua,b Shi-Jin Jianga,b,* a Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Shandong Agricultural University, Shandong Taian, China b Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong Taian, China *Corresponding author at: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Shandong Agricultural University, Shandong Taian, China. Tel.: +86 38 8245799; fax: +86 38 8245799 E-mail address: [email protected] (S-J. Jiang) 1
These authors contributed equally to this work. 16 February 2012
Contents lists available at SciVerse ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Letter to the Editor Detection of aminoglycoside resistance genes in Riemerella anatipestifer isolated from ducks Dear Editor, Riemerella anatipestifer (RA) is the causative agent of a contagious septicaemic disease especially in ducklings, turkeys and other birds. The disease accounts for serious economic losses to the duck industry due to high morbidity and mortality, weight loss and carcase condemnations. Currently, various antibiotics are used to prevent and control RA infection in ducks, but this accelerates the emergence of drug-resistant strains. The resistance of RA to many antibiotics has increased greatly, and some antibiotic resistance genes in RA have been detected (Chang et al., 2003; Zhong et al., 2009; Chen et al., 2010, 2011; Yu et al., 2008; Zheng et al., 2011). Aminoglycoside antibiotics play an important role in the therapy of infections caused by Gram-positive and Gram-negative bacteria. The mechanisms of bacterial resistance to aminoglycosides have been the subject of numerous genetic and biochemical studies (Vakulenko and Mobashery, 2003; Wright, 2005). At present, there are three general mechanisms of aminoglycoside resistance, and enzymatic modification is the most important mechanism (Shaw et al., 1993; Hotta et al., 1996; Hayashi et al., 1997), resulting in a loss of antibacterial activity due to a diminished affinity for the ribosomal A-site target (Llano-Sotelo et al., 2002). These enzymes are assigned to three classes: aminoglycoside nucleotidyltransferases (ant), aminoglycoside acetyltransferases (aac), and aminoglycoside phosphotransferases (aph).These enzymes, including a large number of aminoglycoside-modifying enzymes, are most relevant to clinical resistance in Grampositive organisms (McKay et al., 1996). In this study, the aminoglycoside resistance genes (ant(300 )-Ia, aac(60 )-Ib and aph(30 )-IIa gene) were detected in 38 RA strains isolated from sick ducks in China during April 2005 to October 2011. According to the sequences of aminoglycoside antibiotics resistant genes in E. coli, the following primers were used for the detection of ant(300 )-Ia gene (F-primer, 50 -ATC TGGCTATCTTGCTGACA-30 and R-primer, 50 -TATGACGGGCTGATACTGG-30 ), aac(60 )-Ib gene (F-primer, 50 -ATGACCTTGCGATGCTCTATGA-30 and R-primer, 50 -CGAATGCCTGGCGTGTTT-30 ), and aph(30 )-IIa gene (F-primer, 50 -TGACTGGGCACAACAGACA-30 and R-primer, 50 -TCAAGAAGGCGATAGAAGGC-30 ) (Sun et al., 2011). The amplification program 0378-1135/$ – see front matter ß 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2012.02.027
consisted of an initial step of 10 min at 94 8C, followed by 32 cycles of 50 s at 94 8C, 45 s at 54 8C and 50 s at 72 8C, and a final extension of 10 min at 72 8C. The PCR amplification products were analyzed by 0.8% agarose gel electrophoresis and stained with ethidium bromide. All the positive PCR products were cloned and sequenced, and the nucleotide sequences analyzed by the Clustal W method with DNAstar megalign software (DNAStar Inc., Madison, WI, USA). The aminoglycoside resistance genes were found in all the 38 RA strains, with 36 (95.43%) strains carrying two or three aminoglycoside resistance genes and 2 strains carrying only one aminoglycoside resistance gene (Table 1). A total of 12 (31.58%), 34 (89.47%) and 36 (97.37%) isolates were positive for ant(300 )-Ia, aac(60 )-Ib and aph(30 )-IIa gene, respectively. The nucleotide identity within RA strains was 95.8-100, 97.9-100 and 98.7-100% in ant(300 )-Ia, aac(60 )-Ib and aph(30 )-IIa, respectively. A similar result was also obtained from nucleotide identity between RA and E. coli or Salmonella strains, which was 94.4–100, 98.4–100 and 98.9– 100% in the three genes, respectively. RA, E. coli and Salmonella are common bacterial pathogens in intensive duck husbandry. It is interesting that the three bacteria possess highly related aminoglycoside resistance genes (94.4–100% nucleotide identity). Concluding, ant(300 )-Ia, aac(60 )-Ib and aph(30 )-IIa genes encoding aminoglycoside-modifying enzymes were highly prevalent in RA strains isolated from China, and the three genes had high nucleotide identities with those in E. coIi and Salmonella. These findings highlight the need for further comprehensive study to better understand the mechanisms of aminoglycoside resistance in RA. These newly identified sequences were deposited into the GenBank, with accession numbers JQ664600–JQ664611 Table 1 The detection result of aminoglycoside resistance genes in 38 RA strains. Aminoglycoside resistance genes Negative One resistance gene Two resistance genes Three resistance genes
ant(300 )-Ia aac(60 )-Ib aph(30 )-IIa ant(300 )-Ia + aac(60 )-Ib ant(300 )-Ia + aph(30 )-IIa aac(60 )-Ib + aph(30 )-IIa ant(300 )-Ia + aac(60 )-Ib + aph(30 )-IIa
No. of positive strains
Relevance ratio (%)
0 0 1 1 1 3 24 8
0 0 2.63 2.63 2.63 7.89 63.16 21.05
452
Letter to the Editor / Veterinary Microbiology 158 (2012) 451–452 grons in clinical strains of Riemerella anatipestifer. Vet. Microbiol., doi:10.1016/j.vetmic.2011.11.002.
of ant(300 )-Ia gene, JQ664612–JQ664645 of aac(60 )-Ib gene and JQ664646–JQ664681 of aph(30 )-IIa gene. References
Fang-Fang Yanga,b,1 Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Shandong Agricultural University, Shandong Taian, China b Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong Taian, China a
Chang, C.F., Lin, W.H., Yeh, T.M., Chiang, T.S., Chang, Y.F., 2003. Antimicrobial susceptibility of Riemerella anatipestifer isolated from ducks and the efficacy of ceftiofur treatment. J. Vet. Diagn. Invest. 15, 26–29. Chen, Y.P., Tsao, M.Y., Lee, S.H., Chou, C.H., Tsai, H.J., 2010. Prevalence and molecular characterization of chloramphenicol resistance in Riemerella anatipestifer isolated from ducks and geese in Taiwan. Avian Pathol. 39, 333–338. Chen, Y.P., Lee, S.H., Chou, C.H., Tsai, H.J., 2011. Detection of florfenicol resistance genes in Riemerella anatipestifer isolated from ducks and geese. Vet. Microbiol. 154, 325–331. Hayashi, S.F., Norcia, L.J., Seibel, S.B., Silvia, A.M., 1997. Structure–activity relationships of hygromycin A and its analogs: proteinsynthesis inhibition activity in a cell free system. J. Antibiot. 50, 514–521. Hotta, K., Zhu, C.B., Ogata, T., Sunada, A., Ishikawa, J., Mizuno, S., Kondo, S., 1996. Enzymatic 2-N-acetylation of arbekacin and antibiotic activity of its product. J. Antibiot. 49, 458–464. Llano-Sotelo, B., Azucena Jr., E.F., Kotra, L.P., Mobashery, S., Chow, C.S., 2002. Aminoglycosides modified by resistance enzymes display diminished binding to the bacterial ribosomal aminoacyl-tRNA site. Chem. Biol. 9, 455–463. McKay, G.A., Roestamadji, J., Mobashery, S., Wright, G.D., 1996. Recognition of aminoglycoside antibiotics by the enterococcal–staphylococcal aminoglycoside 30 -phosphotransferase type IIIa: role of substrate amino groups. Antimicrob. Agents Chemother. 40, 2648–2650. Shaw, K.J., Rather, P.N., Hare, R.S., Miller, G.H., 1993. Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes. Microbiol. Rev. 57, 138–163. Sun, H., Lei, Z., Zou, J.F., Wei, Z., Wang, X., Xie, Z.J., Jiang, S.J., 2011. Mechanism of resistance and detection of resistance genes to aminoglycoside among avian Escherichia coli strains from Shandong Province. Chin. J. Vet. Sci. 31, 1279–1282. Vakulenko, S.B., Mobashery, S., 2003. Versatility of aminoglycosides and prospects for their future. Clin. Microbiol. Rev. 16, 430–450. Wright, G.D., 2005. Bacterial resistance to antibiotics: enzymatic degradation and modification. Adv. Drug Deliv. Rev. 57, 1451–1470. Yu, C.Y., Liu, Y.W., Chou, S.J., Chao, M.R., Weng, B.C., Tsay, J.G., Chiu, C.H., Ching Wu, C., Long Lin, T., Chang, C.C., Chu, C., 2008. Genomic diversity and molecular differentiation of Riemerella anatipestifer associated with eight outbreaks in five farms. Avian Pathol. 37, 273–279. Zhong, C.Y., Cheng, A.C., Wang, M.S., Zhu de, K., Luo, Q.H., Zhong, C.D., Li, L., Duan, Z., 2009. Antibiotic susceptibility of Riemerella anatipestifer field isolates. Avian Dis. 53, 601–607. Zheng, F., Lin, G., Zhou, J., Cao, X., Gong, X., Wang, G., Qiu, C., 2011. Discovery and characterization of gene cassettes-containing inte-
Ya-Ni Sun1 College of Veterinary Medicine, Northwest A & F University, Shanxi Yangling, China Jing-Xin Lia,b Hui Wanga,b Meng-Jiao Zhaoa,b Jing Sua,b Zhen-Jun Zhanga,b Hong-Jie Liua,b Shi-Jin Jianga,b,* a Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Shandong Agricultural University, Shandong Taian, China b Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong Taian, China *Corresponding author at: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Shandong Agricultural University, Shandong Taian, China. Tel.: +86 38 8245799; fax: +86 38 8245799 E-mail address: [email protected] (S-J. Jiang) 1
These authors contributed equally to this work. 16 February 2012