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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 2011, p. 373–375 Copyright 2011, American Society for Microbiology. All Rights Reserved.
Novel Apramycin Resistance Gene apmA in Bovine and Porcine Methicillin-Resistant Staphylococcus aureus ST398 Isolatesᰔ Andrea T. Feßler, Kristina Kadlec, and Stefan Schwarz* Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, Ho ¨ltystr. 10, 31535 Neustadt-Mariensee, Germany Received 13 August 2010/Accepted 17 September 2010 A novel apramycin resistance gene, apmA, was detected on the ca.-40-kb resistance plasmid pAFS11 from
bovine methicillin-resistant Staphylococcus aureus (MRSA) of sequence type 398 (ST398). The apmA gene coded
for a protein of 274 amino acids that was related only distantly to acetyltransferases involved in chloram-
phenicol or streptogramin A resistance. NsiI deletion of apmA resulted in a 16- to 32-fold decrease in the
apramycin MICs. An apmA-specific PCR identified this gene in one additional bovine and four porcine MRSA
ST398 isolates.
Methicillin-resistant Staphylococcus aureus (MRSA) of se- Laboratory Standards Institute (CLSI) are currently available quence type 398 (ST398) has been identified mainly as a col- (4). One of these isolates, the bovine MRSA isolate 11, was onizer of the skin and the mucosal surfaces of swine (6, 26, 28), chosen for further analysis of the genetic basis of apramycin although, more rarely, such isolates have also been found to be resistance. The bovine MRSA isolate 11 carried a staphylococ- involved in infections of swine (9, 17, 18, 24). Moreover, cal cassette chromosome mec element of type V (SCCmec V) MRSA ST398 has also been detected in other animals, such as and displayed the multilocus sequence type (MLST) ST398, cattle (8, 23), horses (25, 27), poultry (15), dogs (16), and rats the spa type t2576, and the dru type dt11a (8). Plasmid analysis (22), and in humans with exposure to MRSA ST398-colonized identified the ca.-40-kb plasmid pAFS11, which, upon trans- animals (7, 20, 28, 30). As a colonizer, MRSA ST398 is subject formation into S. aureus RN4220, mediated a multiresistance to selective pressure by antimicrobial agents that are not used phenotype (Table 1). The corresponding resistance genes were primarily to control staphylococcal infections and, as a conse- detected by specific PCR assays (8, 9, 14). In addition to quence, may acquire novel or uncommon resistance genes.
kanamycin and neomycin resistance via aadD, macrolide-lin- One such example is provided by the observation that chlor- cosamide-streptogramin B resistance via erm(B), tetracycline amphenicol-resistant MRSA ST398 isolates from swine (9) and resistance via tet(L), and trimethoprim resistance via dfrK, cattle (8) did not carry any of the usually found staphylococcal plasmid pAFS11 conferred a high apramycin MIC of Ն128 cat genes for chloramphenicol resistance (19) but harbored the ␮g/ml. The S. aureus RN4220 transformant carrying pAFS11, phenicol exporter gene fexA, which also confers resistance to however, was classified as intermediate to gentamicin (MIC of florfenicol. Florfenicol is a fluorinated chloramphenicol deriv- ative that is widely used for the control of respiratory tract An 11,312-bp EcoRI fragment of pAFS11 was cloned into infections in cattle and swine. Another example is apramycin pBluescript II SKϩ (Stratagene). Recombinant plasmids were transformed into E. coli strain JM101, and transfor- Apramycin is an aminocyclitol antibiotic that is used exclu- mants were selected on apramycin-supplemented Luria-Ber- sively in veterinary medicine for the treatment of Escherichia tani agar (15 ␮g/ml). Sequence analysis was conducted by coli infections in swine, cattle, sheep, poultry, or rabbits. Stud- primer walking starting with M13 universal and reverse ies of apramycin-resistant Enterobacteriaceae identified the primers. A schematic representation of the seven reading gene aac(3)-IV, which is located mostly on plasmids and con- frames found on this EcoRI fragment is shown in Fig. 1.
fers resistance to apramycin and gentamicin (2, 3, 5, 21, 29). In This segment comprised part of a Tn917 transposon with contrast to the wealth of data available for apramycin resis- one terminal repeat and the entire erm(B) gene. A reading tance in Enterobacteriaceae (1, 31, 32), no information about frame for a 315-amino-acid (aa) protein with 30.9 and 31.4% apramycin resistance in staphylococci exists.
identity to distinctly larger chromosome replication initia- During two survey studies on MRSA ST398 from diseased swine and dairy cattle, 4/54 porcine and 2/16 bovine isolates hominis (NCBI accession no. ZP_04059882) and Staphylo- revealed high apramycin MIC values of Ն32 ␮g/ml (8, 9).
coccus warneri (NCBI accession no. ZP_04678490), respec- These isolates were tentatively classified as resistant, although tively, was detected. Further downstream was the reading no clinical breakpoints for apramycin approved by the Clinical frame for a 263-aa ParA protein that corresponded closely(96.2 and 95.1% identity, respectively) to the chromosomepartitioning ATPases of Staphylococcus capitis (NCBI acces- * Corresponding author. Mailing address: Institute of Farm Animal sion no. ZP_03614545) and S. aureus (NCBI accession no.
Genetics, Friedrich-Loeffler-Institut, Ho ACY12632). A complete IS257 element was identified, but Mariensee, Germany. Phone: 49-5034-871-241. Fax: 49-5034-871-246.
this did not exhibit 8-bp direct repeat sequences in the up- ᰔ Published ahead of print on 27 September 2010.
and downstream segments. The lack of these direct repeats TABLE 1. Comparative analysis of the bovine MRSA ST398 isolate 11, S. aureus RN4220, and the S. aureus RN4220 apmA, erm(B), tet(L), tet(M), tet(K), dfrK, aadD, mecA, blaZ apmA, erm(B), tet(L), dfrK, aadD a APR, apramycin; GEN, gentamicin; ERY, erythromycin; CLI, clindamycin; TET, tetracycline; TMP, trimethoprim; KAN, kanamycin; NEO, neomycin; OXA, suggested that recombination events via this insertion se- TTTC-3Ј) (annealing temperature, 52°C; amplicon size, 656 quence have occurred. A complete reading frame for a bp) was developed and applied to MRSA ST398 isolates.
347-aa protein and the 3Ј end of a reading frame (190 aa) While the remaining bovine and the four porcine apramycin- showed 48.4% and 54.5% identity to IcaC (NCBI accession resistant isolates were positive for apmA, the isolates with no. YP_189846) and IcaB (NCBI accession no. YP_189845), MICs of Յ16 ␮g/ml were negative. All five additional isolates respectively, from a Staphylococcus epidermidis isolate.
harbored SCCmec V and showed the spa type t011 and the dru To confirm the role of the seventh reading frame, designated type dt11a (8, 9). Transfer and hybridization experiments iden- apmA, in apramycin resistance, the EcoRI fragment was di- tified apmA in all five cases on plasmids of ca. 40 kb that were gested with NsiI, which cuts once within the apmA reading indistinguishable from or closely related to pAFS11 in their frame, once within the IS257 sequence, and once within the EcoRI, HindIII, BglII, and PvuI restriction patterns. All of icaB-like gene. Deletion clones in E. coli JM101 were tested for these plasmids also harbored tet(L), dfrK, aadD, and erm(B) in their apramycin MICs by broth microdilution according to the CLSI document M31-A3 (4). In comparison to clones carrying Recent studies on antimicrobial resistance genes in MRSA the original EcoRI fragment, all three deletion clones showed ST398 led to the identification of a number of novel or unusual a 16- to 32-fold decrease in the apramycin MICs and also an resistance genes, such as dfrK (10), vga(C) (11), erm(T) (12), 8-fold decrease in the gentamicin MICs. The apmA gene codes and cfr (13). All of these genes were located on plasmids.
for a 274-aa protein that shows limited similarity to other Analysis of these plasmids suggested that recombination and proteins deposited in the databases. The best matches were cointegrate formation played a major role in the acquisition of 38.1% identity to a VatB-like xenobiotic acetyltransferase pro- novel resistance genes by MRSA ST398. In most of the described plasmids, insertion sequences, such as IS257 or NP_246134) and 33.3% identity to a putative chloramphenicol ISSau10 (10, 12), seemed to be involved in recombination acetyltransferase from Escherichia fergusonii (NCBI accession processes. This is, to the best of our knowledge, the first de- no. YP_002383245). Based on the apmA sequence, a PCR scription of an apramycin resistance gene in Gram-positive assay using the primers apmA-fw (5Ј-CGTTTGCTTCGTGC cocci. The presence of apmA on the multiresistance plasmid ATTAAA-3Ј) and apmA-rev (5Ј-TTGACACGAAGGAGGG pAFS11 enables its persistence and coselection under the se-lective pressure imposed by the use of kanamycin, neomycin,tetracyclines, macrolides, lincosamides, or trimethoprim.
Nucleotide sequence accession number. The nucleotide se-
quence of the 11,312-bp EcoRI fragment of plasmid pAFS11has been deposited in the EMBL database under accessionnumber FN806789.
This study was financially supported by internal funding from the REFERENCES
1. Boerlin, P., R. Travis, C. L. Gyles, R. Reid-Smith, N. Janecko, H. Lim, V.
Nicholson, S. A. McEwen, R. Friendship, and M. Archambault. 2005. Anti-
microbial resistance and virulence genes of Escherichia coli isolates from
swine in Ontario. Appl. Environ. Microbiol. 71:6753–6761.
2. Chaslus-Dancla, E., J. L. Martel, C. Carlier, J. P. Lafont, and P. Courvalin.
1986. Emergence of aminoglycoside 3-N-acetyltransferase IV in Escherichia FIG. 1. Schematic presentation of the seven reading frames found coli and Salmonella typhimurium isolated from animals in France. Antimi- on the 11,312-kb EcoRI fragment of pAFS11. The arrows indicate the crob. Agents Chemother. 29:239–243.
extents and directions of transcription. A distance scale in kb is given 3. Chaslus-Dancla, E., P. Pohl, M. Meurisse, M. Marin, and J. P. Lafont. 1991.
below the map. The IS257 element is shown as a black box, with the High genetic homology between plasmids of human and animal origins white arrow indicating the transposase gene tnp. The MICs of apra- conferring resistance to the aminoglycosides gentamicin and apramycin. An- mycin and gentamicin conferred by the complete EcoRI fragment and timicrob. Agents Chemother. 35:590–593.
the corresponding NsiI deletion clones are shown on the right-hand 4. CLSI. 2008. Performance standards for antimicrobial disk and dilution sus-
ceptibility test for bacteria isolated from animals; approved standard—third APRAMYCIN RESISTANCE GENE apmA IN MRSA ST398 edition. CLSI document M31-A3. Clinical and Laboratory Standards Insti- Staphylococcus aureus and Staphylococcus pseudintermedius detected in the BfT-GermVet monitoring programme 2004–2006 in Germany. J. Antimi- 5. Davies, J., and S. O’Connor. 1978. Enzymatic modification of aminoglyco-
crob. Chemother. 61:282–285.
side antibiotics: 3-N-acetyltransferase with broad specificity that determines 19. Schwarz, S., C. Kehrenberg, B. Doublet, and A. Cloeckaert. 2004. Molecular
resistance to the novel aminoglycoside apramycin. Antimicrob. Agents Che- basis of bacterial resistance to chloramphenicol and florfenicol. FEMS Mi- mother. 14:69–72.
crobiol. Rev. 28:519–542.
6. de Neeling, A. J., M. J. van den Broek, E. C. Spalburg, M. G. van Santen-
20. Smith, T. C., M. J. Male, A. L. Harper, J. S. Kroeger, G. P. Tinkler, E. D.
Verheuvel, W. D. Dam-Deisz, H. C. Boshuizen, A. W. van de Giessen, E. van
Moritz, A. W. Capuano, L. A. Herwaldt, and D. J. Diekema. 2009. Methicil-
Duijkeren, and X. W. Huijsdens. 2007. High prevalence of methicillin resis-
lin-resistant Staphylococcus aureus (MRSA) strain ST398 is present in mid- tant Staphylococcus aureus in pigs. Vet. Microbiol. 122:366–372.
western U.S. swine and swine workers. PLoS One 4:e4258.
7. Denis, O., C. Suetens, M. Hallin, B. Catry, I. Ramboer, M. Dispas, G.
21. Threlfall, E. J., B. Rowe, J. L. Ferguson, and L. R. Ward. 1986. Character-
Willems, B. Gordts, P. Butaye, and M. J. Struelens. 2009. Methicillin-resis-
ization of plasmids conferring resistance to gentamicin and apramycin in tant Staphylococcus aureus ST398 in swine farm personnel, Belgium. Emerg.
strains of Salmonella typhimurium phage type 204c isolated in Britain. J. Hyg.
Infect. Dis. 15:1098–1101.
(Lond.) 97:419–426.
8. Feßler, A., C. Scott, K. Kadlec, R. Ehricht, S. Monecke, and S. Schwarz.
22. van de Giessen, A. W., M. G. van Santen-Verheuvel, P. D. Hengeveld, T.
2010. Characterization of methicillin-resistant Staphylococcus aureus ST398 Bosch, E. M. Broens, and C. B. Reusken. 2009. Occurrence of methicillin-
from cases of bovine mastitis. J. Antimicrob. Chemother. 65:619–625.
resistant Staphylococcus aureus in rats living on pig farms. Prev. Vet. Med.
9. Kadlec, K., R. Ehricht, S. Monecke, U. Steinacker, H. Kaspar, J. Mankertz,
91:270–273.
and S. Schwarz. 2009. Diversity of antimicrobial resistance pheno- and
23. Vanderhaeghen, W., T. Cerpentier, C. Adriaensen, J. Vicca, K. Hermans,
genotypes of methicillin-resistant Staphylococcus aureus ST398 from dis- and P. Butaye. 2010. Methicillin-resistant Staphylococcus aureus (MRSA)
eased swine. J. Antimicrob. Chemother. 64:1156–1164.
ST398 associated with clinical and subclinical mastitis in Belgian cows. Vet.
10. Kadlec, K., and S. Schwarz. 2009. Identification of a novel trimethoprim
Microbiol. 144:166–171.
resistance gene, dfrK, in a methicillin-resistant Staphylococcus aureus ST398 24. van Duijkeren, E., M. D. Jansen, S. C. Flemming, H. de Neeling, J. A.
strain and its physical linkage to the tetracycline resistance gene tet(L).
Wagenaar, A. H. W. Schoormans, A. van Nes, and A. C. Fluit. 2007. Methi-
Antimicrob. Agents Chemother. 53:776–778.
cillin-resistant Staphylococcus aureus in pigs with exudative epidermitis.
11. Kadlec, K., and S. Schwarz. 2009. Identification of a novel ABC transporter
Emerg. Infect. Dis. 13:1408–1410.
gene, vga(C), located on a multiresistance plasmid from a porcine methicil- 25. van Duijkeren, E., M. Moleman, M. M. Sloet van Oldruitenborgh-Ooster-
lin-resistant Staphylococcus aureus ST398 strain. Antimicrob. Agents Che- baan, J. Multem, A. Troelstra, A. C. Fluit, W. J. van Wamel, D. J. Houwers,
mother. 53:3589–3591.
A. J. de Neeling, and J. A. Wagenaar. 2010. Methicillin-resistant Staphylo-
12. Kadlec, K., and S. Schwarz. 2010. Identification of a plasmid-borne resis-
coccus aureus in horses and horse personnel: an investigation of several tance gene cluster comprising the resistance genes erm(T), dfrK, and tet(L) in outbreaks. Vet. Microbiol. 141:96–102.
a porcine methicillin-resistant Staphylococcus aureus ST398 strain. Antimi- 26. Voss, A., F. Loeffen, J. Bakker, C. Klaassen, and M. Wulf. 2005. Methicillin-
crob. Agents Chemother. 54:915–918.
resistant Staphylococcus aureus in pig farming. Emerg. Infect. Dis. 11:1965–
13. Kehrenberg, C., C. Cuny, B. Strommenger, S. Schwarz, and W. Witte. 2009.
Methicillin-resistant and -susceptible Staphylococcus aureus strains of clonal 27. Walther, B., S. Monecke, C. Ruscher, A. W. Friedrich, R. Ehricht, P. Slick-
lineages ST398 and ST9 from swine carry the multidrug resistance gene cfr.
ers, A. Soba, C. G. Wleklinski, L. H. Wieler, and A. Lu
¨bke-Becker. 2009.
Antimicrob. Agents Chemother. 53:779–781.
Comparative molecular analysis substantiates zoonotic potential of equine ¨thje, P., and S. Schwarz. 2006. Antimicrobial resistance of coagulase-
methicillin-resistant Staphylococcus aureus. J. Clin. Microbiol. 47:704–710.
negative staphylococci from bovine subclinical mastitis with particular ref- 28. Witte, W., B. Strommenger, C. Stanek, and C. Cuny. 2007. Methicillin-
erence to macrolide-lincosamide resistance phenotypes and genotypes. J.
resistant Staphylococcus aureus ST398 in humans and animals, Central Eu- Antimicrob. Chemother. 57:966–969.
rope. Emerg. Infect. Dis. 13:255–258.
15. Nemati, M., K. Hermans, U. Lipinska, O. Denis, A. Deplano, M. Struelens,
29. Wray, C., R. W. Hedges, K. P. Shannon, and D. E. Bradley. 1986. Apramycin
L. A. Devriese, F. Pasmans, and F. Haesebrouck. 2008. Antimicrobial resis-
and gentamicin resistance in Escherichia coli and salmonellas isolated from tance of old and recent Staphylococcus aureus isolates from poultry: first farm animals. J. Hyg. (Lond.) 97:445–456.
detection of livestock-associated methicillin-resistant strain ST398. Antimi- 30. Wulf, M. W., E. Tiemersma, J. Kluytmans, D. Bogaers, A. C. Leenders,
crob. Agents Chemother. 52:3817–3819.
M. W. Jansen, J. Berkhout, E. Ruijters, D. Haverkate, M. Isken, and A. Voss.
16. Nienhoff, U., K. Kadlec, I. F. Chaberny, J. Verspohl, G.-F. Gerlach, S.
2008. MRSA carriage in healthcare personnel in contact with farm animals.
Schwarz, D. Simon, and I. Nolte. 2009. Transmission of methicillin-resistant
J. Hosp. Infect. 70:186–190.
Staphylococcus aureus strains between humans and dogs: two case reports. J.
31. Yates, C. M., M. C. Pearce, M. E. Woolhouse, and S. G. Amyes. 2004. High
Antimicrob. Chemother. 64:660–662.
frequency transfer and horizontal spread of apramycin resistance in calf 17. Pomba, C., F. Baptista, N. Couto, F. Louc
˜o, and H. Hasman. 2010. Methi-
faecal Escherichia coli. J. Antimicrob. Chemother. 54:534–537.
cillin-resistant Staphylococcus aureus CC398 isolates with indistinguishable 32. Zhang, X. Y., L. J. Ding, and M. Z. Fan. 2009. Resistance patterns and
ApaI restriction patterns in colonized and infected pigs and humans. J.
detection of aac(3)-IV gene in apramycin-resistant Escherichia coli isolated Antimicrob. Chemother. 65:2479–2481.
from farm animals and farm workers in northeastern of China. Res. Vet. Sci.
18. Schwarz, S., K. Kadlec, and B. Strommenger. 2008. Methicillin-resistant
87:449–454.

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