ABSTRACT
The multiresistance gene cfr was found in 8/231 porcine methicillin-resistant Staphylococcus aureus isolates. They were characterized by multilocus sequence typing, spa typing, dru typing, and staphylococcal cassette chromosome mec (SCCmec) typing as ST627-t002-dt12w-IVb, ST6-t304-dt12w-IVb, ST9-t899-dt12w-IVb, ST9-t899-dt12ae-IVb, or ST63-t899-dt12v-IVb. Different cfr gene regions were detected on plasmids of ca. 35 kb in seven isolates. For the first time, an ISEnfa4-cfr-IS256 fragment was found to be inserted upstream of the ccr genes in a chromosomal SCCmec IVb element of the remaining isolate.
TEXT
Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) strains mainly play a role as colonizers of food-producing animals and humans who have occupational and otherwise close contact with these animals (1–5). However, they can also cause infections in humans (6) and animals (7, 8). The important LA-MRSA clones are usually of the multilocus sequence type 398 (ST398) in the United States (1, 2) and European countries (9) and/or of ST9 in Asian countries (10), including China. LA-MRSA strains have acquired a number of novel and unusual antimicrobial resistance genes (11–15), including multiresistance genes that confer resistance to critically and highly important antimicrobial agents in human medicine (16). One such gene is the gene cfr, which confers resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A (17). Oxazolidinones are last resort antimicrobial agents for the control of serious infections caused by MRSA and vancomycin-resistant enterococci in humans. Although cfr-carrying MRSA strains have been reported in human medicine (18, 19), cfr-carrying LA-MRSA strains of animal origin have been reported rarely (20, 21).
The 231 porcine MRSA isolates included in this study were collected in August 2012 and August 2013 from nasal swabs of healthy pigs at farms (n = 7) and slaughterhouses (n = 3) in the provinces of Henan (n = 119) and Shandong (n = 45) and in Shanghai (n = 15) and from the lungs of diseased pigs at an animal hospital in the Guangdong province (n = 52). All isolates were screened for the presence of cfr using previously described primers (22). Only eight (3.5%) of the MRSA isolates were positive for cfr. All cfr-carrying MRSA isolates had linezolid MICs of 4 mg/liter, which classifies them as borderline susceptible based on the current CLSI-approved breakpoints (23). cfr-positive staphylococci and enterococci with linezolid MICs of 4 mg/liter have been reported before (24, 25). The carriage rates for cfr differed geographically, with 5/15 (33.3%) cfr-positive isolates observed in Shanghai, followed by 2/52 (3.84%) in the Guangdong Province and 1/119 (0.84%) in the Henan Province. None of the isolates from Shandong contained cfr.
The eight cfr-carrying isolates were subjected to staphylococcal chromosomal cassette mec (SCCmec) typing, multilocus sequence typing (MLST) (http://saureus.mlst.net), spa typing (http://spaserver.ridom.de), dru typing (http://dru-typing.org), and SmaI pulsed-field gel electrophoresis (PFGE) (4). All isolates shared SCCmec type IVb, while three different PFGE-MLST-spa profiles were observed: A-ST6-t304, B-ST627-t002, and C-ST63-t899 (Table 1). ST63 is a variant of ST9, differing by three single base pair exchanges in the arcC locus. The dru type dt12w was observed in six strains, while dt12v and the novel dru type dt12ae were observed in individual lung isolates collected in Guangdong.
Characteristics of the cfr-carrying MRSA isolates from pigs at slaughter and from the lungs of diseased pigs
All eight MRSA isolates were examined for their antimicrobial resistance phenotypes and genotypes. Resistance genes were detected by specific PCR assays (22, 26, 27) and sequence analyses. Antimicrobial susceptibility testing was performed by broth microdilution (28, 29), and 16 antimicrobial agents, including ampicillin, penicillin G, cefoxitin, oxacillin, chloramphenicol, florfenicol, erythromycin, tiamulin, clindamycin, tetracycline, streptomycin, virginiamycin M1, linezolid, trimethoprim-sulfamethoxazole, spectinomycin, and vancomycin, were used. Besides cfr, all isolates harbored the mecA and fexA genes for resistance to methicillin and chloramphenicol-florfenicol, respectively, and all isolates, except HP15 and HP11, carried the tetracycline resistance gene tet(L). Three isolates (HP29, HP30, and HP32) contained the gene vga(A)V, while another three isolates (SH50, 1518, and 1530) carried the gene lsa(E), each of which code for ABC transporters that confer resistance to lincosamides, pleuromutilins, and streptogramin A. Isolates SH50, 1518, and 1530 also harbored a chromosomal 12,120-bp segment carrying a resistance gene cluster with the genes aadE (streptomycin resistance), spw (spectinomycin resistance), lsa(E), and lun(B) (lincosamide resistance). This multiresistance gene cluster showed 99.7% nucleotide sequence identity to that found on a 250-kb plasmid from MRSA strain C2944 (26).
A 4,380-bp segment, including orf6-erm(33)-spc-tnpC-tnpB-orf11 in isolates 1518 and 1530, showed 99.8% nucleotide sequence identity to the corresponding region of plasmid pSCFS1 from Staphylococcus sciuri (GenBank accession no. AJ579365) (30). The gene erm(33), which mediates resistance to macrolides, lincosamides, and streptogramin B antibiotics and represents an in vivo recombination product between erm(A) and erm(C) (31), has so far only been detected on the multiresistance plasmid pSCFS1 from bovine S. sciuri (30).
S1-nuclease PFGE mapping and Southern blot analysis (32) revealed that the gene cfr was located on ∼35-kb plasmids in seven isolates and in the chromosomal DNA of isolate 1518 (data not shown). The flanking regions of cfr in all isolates were determined using whole genome sequencing and a modified random primer walking strategy (33). Plasmid pSS-02, carrying the intact insertion sequence IS21-558 located immediately upstream of cfr, was detected in the five isolates from Shanghai and in one isolate from Guangdong (Fig. 1). The cfr region of this plasmid was similar to that of plasmid pSCFS3, which has been detected in staphylococci from China, the United States, and Europe (22, 34–36). A pSCFS7-like plasmid, which contained a cfr region similar to that of plasmid pSCFS7 (37), was found in isolate SH50 from Henan (Fig. 1).
Schematic representation of the organization of the cfr flanking regions in different plasmids identified in this study as well as of the novel location of cfr in an SCCmec complex. Gray-shaded areas represent regions of >99.9% nucleotide sequence identity. The arrows indicate the positions and orientations of the genes with the arrowhead giving the direction of transcription. Insertion sequences are shown as boxes with the arrow inside the box symbolizing the respective transposase gene(s). The designation hp indicates genes coding for hypothetical proteins. The arrows 1F to 7F and 1R to 7R indicate the positions of the forward and reverse primers 1 to 7.
In the 26,737-bp contig of chromosomal DNA of MRSA isolate 1518, a 5,327-bp element was detected, which included a cfr-carrying central region (1,050 bp) flanked by a copy of ISEnfa4 and a copy of IS256 located in the same orientation. This region showed 97.1% (5,172/5,327 bp) nucleotide sequence identity to that of plasmid pSS-01 (GenBank accession no. JQ041372). ISEnfa4, which was previously named IS256-like in pSS-01, harbored a 1,324-bp transposase gene that exhibited 88.9% (1,177/1,324 bp) nucleotide sequence identity to that of IS256. This ISEnfa4-cfr-IS256 segment was inserted into the hp1 gene located in the joining (J) region 1 of a type IVb SCCmec element. This SCCmec element consisted of (i) a variant of a type 1 cassette chromosome recombinase (ccr) gene complex composed of a truncated ccrA1 gene and an intact ccrB1 gene, (ii) a class D mec gene complex (IS431-mecA-ΔmecR1), and (iii) J regions J1, J2, and J3 flanking the ccr and mec complexes (Fig. 1). Seven sets of PCR primers (1-F/R to 7-F/R) (see Table S1 in the supplemental material) were used to confirm the order of these genes in the chromosomal DNA of isolate 1518 (Fig. 1). The truncated hp1 gene, in which a 339-bp deletion resulted from the integration of ISEnfa4-cfr-IS256, exhibited 99.0% nucleotide sequence identity to the corresponding region of the 1,428-bp hp1 gene in the J1 region of MRSA JCSC6690 (GenBank accession no. AB705453). It should be noted that the class D mec gene complex has previously only been observed in methicillin-resistant Staphylococcus caprae (38).
In conclusion, to the best of our knowledge, this is the first time that cfr was detected in the J region of an SCCmec element. The finding that cfr-carrying MRSA isolates from pigs carry additional resistance genes warrants continuous surveillance, as it underlines the potential for coselection and persistence of these isolates under the selective pressure by various other antimicrobial agents.
Nucleotide sequence accession number.The DNA sequence of the cfr-carrying segment in MRSA isolate 1518 was assigned GenBank accession no. KP777553.
ACKNOWLEDGMENTS
This work was funded by the National Natural Science Foundation of China (grant 31472237) and also by grant 01KI1301D (MedVet-Staph 2) of the German Federal Ministry of Education and Research (BMBF) provided through the German Aerospace Center (DLR).
FOOTNOTES
- Received 4 March 2015.
- Returned for modification 12 March 2015.
- Accepted 25 March 2015.
- Accepted manuscript posted online 30 March 2015.
Supplemental material for this article may be found at http://dx.doi.org/10.1128/AAC.00500-15.
- Copyright © 2015, American Society for Microbiology. All Rights Reserved.
