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.
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REPORT FOR PHARMACIES. Intensive use of disinfectants and antibiotics has led to the emergence of many resistant microorganisms that are increasingly becoming the cause of various infections. Especially Methicillin Resistant Staphylococcus Aureus (MRSA) and Clostridium is a serious problem even for hospitals now causing severe illnesses and death among patients. Currently used process

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