Analysis of Resistance to Florfenicol and the Related Mechanism of Dissemination in Different Animal-Derived Bacteria

Abstract

Bacterial resistance to antibiotics has become an important concern for public health. This study was aimed to investigate the characteristics and the distribution of the florfenicol-related resistance genes in bacteria isolated from four farms. A total of 106 florfenicol-resistant Gram-negative bacilli were examined for florfenicol-related resistance genes, and the positive isolates were further characterized. The antimicrobial sensitivity results showed that most of them (100, 94.33%) belonged to multidrug resistance Enterobacteriaceae. About 91.51% of the strains carried floR gene, while 4.72% carried cfr gene. According to the pulsed-field gel electrophoresis results, 34 Escherichia coli were subdivided into 22 profiles, the genetic similarity coefficient of which ranged from 80.3 to 98.0%. The multilocus sequence typing (MLST) results revealed 17 sequence types (STs), with ST10 being the most prevalent. The genome sequencing result showed that the Proteus vulgaris G32 genome consists of a 4.06-Mb chromosome, a 177,911-bp plasmid (pG32-177), and a 51,686-bp plasmid (pG32-51). A floR located in a drug-resistant region on the chromosome of P. vulgaris G32 was with IS91 family transposase, and the other floR gene on the plasmid pG32-177 was with an ISCR2 insertion sequence. The cfr gene was located on the pG32-51 flanked by IS26 element and TnpA26. This study suggested that the mobile genetic elements played an important role in the replication of resistance genes and the horizontal resistance gene transfer.

Keywords: MLST; PFGE; cfr; floR; flofenicol; genomics.

Figure 1

Pulsed-field gel electrophoresis (PFGE) pattern of 34 E. coli resistant to more than seven drugs. The chromosomal DNA of 34 E. coli isolates carrying the floR genes were digested with the restriction enzyme XbaI and then subjected to PFGE analysis.

Figure 2

EBURST result of 6,475 sequence types (STs) in Pubmlst/E. coli database. All the 6,475 STs were clustered into 17 STs.

Figure 3

The circular map of the pG32-51 genome. Counting from outside toward the center, the first circle refers to the position in base pairs. The second circle consists of two-direction arrows which indicate the position of the gene and the marked genes encoded on the leading strand (outwards) or lagging strand (inwards). The different function genes are shown in different colors: red, drug-resistance related genes; purple, conjugation and transfer; green and yellow, transposase/insertion sequences; rose red, replication; blue, recominase; silver, unknown function genes. The third circle shows the GC skew (G–C/G+C), with a positive GC skew toward the outside and a negative GC skew toward the inside. The fourth circle shows the GC content with an average of 50%, whereby a G+C content of more than 50% is shown toward the outside and a G+C content of <50% toward the inside.

Figure 4

Genome structure comparison of P. vulgaris G32 with CYPV1 and FDAARGOS_366. The corresponding blocks from these three strains are shown according to the gene content or sequence similarities. The blocks below the line in strains CYPV1 and FDAARGOS_366 genome indicated that the sequence in the block was in a reverse direction compared to the corresponding region in P. vulgaris G32. The denser and higher lines represented more genes and higher similarities between the sequences.

Figure 5

Comparison of the floR gene regions on the chromosome of P. vulgaris G32, Proteus CYPV1 (accession number CP012675), Proteus FDAARGOS-366 (accession number CP023965), and V. cholerae HC1037 (accession number CP026647.1). The homolog genes are marked with the same color and lined together, respectively.

Figure 6

Comparison of the floR gene regions on the pG32-177, the sequenced plasmids of E. coli YJMC8 (accession number KY019259.1) and E. coli SHP45 (accession number KU341381.1).

Figure 7

Comparison of the cfr gene regions on pG32-51, E. coli 8ZG12D (accession number KY865320.1), E. coli SH21G (accession number KJ453115.1), and the sequences of P. vulgaris strain PV-01 (accession number JF969273.1).