Characterization of a novel small plasmid carrying the florfenicol resistance gene floR in Haemophilus parasuis.

Sir, The Gram-negative bacterium Haemophilus parasuis is the causative agent of Glässer’s disease, which is characterized by various combinations of meningoencephalitis, polyserositis, polyarthritis and bacterial pneumonia, resulting in major economic losses to the swine industry worldwide. Florfenicol is used exclusively in veterinary medicine and was approved for the treatment of porcine respiratory diseases in China in 1999. So far, five florfenicol resistance genes (floR, fexA, fexB, cfr and optrA) have been reported in bacteria of animal origin. In Gram-negative bacteria, the floR gene is the most important contributor to florfenicol resistance and has been described in various species. To date, studies on the antimicrobial susceptibility of H. parasuis have been carried out in several countries (China, Denmark, the UK, Spain, the Czech Republic and Australia), which reported that all of the clinical isolates were susceptible to florfenicol. Here, we report the emergence of florfenicol-resistant H. parasuis isolates in China, which is attributable to a novel small plasmid bearing the floR gene. A total of 62 H. parasuis isolates were collected from pigs with respiratory diseases in Shanghai and Jiangsu province, China, from March 2013 to May 2014. Conventional biochemical testing, diagnostic PCR analysis and 16S rDNA sequencing were used to identify the isolates. Since there is no standard method for susceptibility testing of H. parasuis, broth microdilution was performed according to the recommendations of the CLSI (VET01-A4, 2013) for Actinobacillus pleuropneumoniae. A. pleuropneumoniae ATCC 27090 served as the quality control strain. Of the 62 H. parasuis isolates, 3 (ASB6W, ASB17W and A4) showed high florfenicol MIC values of 8 mg/L, while the remaining 59 strains exhibited low MIC values of florfenicol, ranging from 0.25 to 1 mg/L. The isolates ASB6W and ASB17W were obtained from a farm in Jiangsu province and the A4 isolate was from a farm in Shanghai. The three isolates were tentatively considered as florfenicol resistant according to the distribution of MIC values of the 62 H. parasuis isolates and the CLSI breakpoint of florfenicol for A. pleuropneumoniae. The presence of the floR gene in the three strains was confirmed by PCR with the primers floRF (5′-GCGATATTCATTACTTTGGC-3′) and floRR (5′-TAGGATGAAGG TGAGGAATG-3′) and subsequent sequencing of the amplicon. Plasmid DNA was extracted using the Qiagen Plasmid Mini Kit (Qiagen, Hilden, Germany) and profiling analysis revealed that each of the three florfenicol-resistant H. parasuis isolates harboured only a single plasmid of 6 kb. The aforementioned PCR fragment of the floR gene was labelled using the DIG High Prime DNA Labelling and Detection Kit (Roche Diagnostics, Mannheim, Germany). Southern blot analysis performed with the floR-specific probe showed that the floR gene was located on the 6 kb plasmids in the three isolates (data not shown). Subsequently, the three plasmids from the three isolates were completely sequenced by primer walking, starting with the floR-specific primers described above. Sequence comparisons revealed that the three plasmids were identical. This plasmid, designated as pHPSF1, was transformed into the plasmid-free H. parasuis isolate D20 by electrotransformation. The transformant exhibited elevated MICs of florfenicol (0.5–8 mg/L) and chloramphenicol (0.5–8 mg/L) when compared with those of the recipient strain. These results suggested that the plasmid pHPSF1 carried the floR gene and was responsible for florfenicol resistance in the three H. parasuis isolates. The plasmid pHPSF1 was 6328 bp (GenBank accession number KR262062) and consisted of five ORFs encoding the florfenicol resistance protein FloR, which consists of 404 amino acids, the 109 amino acid putative transcriptional regulator LysR, a potential Rep protein of 337 amino acids involved in plasmid replication and two Mob proteins associated with plasmid mobilization (MobC of 97 amino acids and MobA/L of 450 amino acids). The FloR protein of plasmid pHPSF1 had a typical size of 404 amino acids and 12 putative transmembrane domains, which is same as other functionally active members of the FloR family. It showed amino acid identity of 88.4% (to the FloR variant of Stenotrophomonas maltophilia, accession number AIU94575) to 97.5% (to the FloR of Escherichia coli, accession number CEL26452) with the FloR proteins in the GenBank database. Database searches identified three plasmids that showed high sequence homology with pHPSF1, including the 5486 bp plasmid p11745 carrying the tet(B) tetracycline resistance gene (GenBank accession number DQ176855) of A. pleuropneumoniae isolated from a pig in Spain, the 10 874 bp floR-carrying plasmid