Inactivation of chloramphenicol and florfenicol by a novel chloramphenicol hydrolase

Abstract

Chloramphenicol and florfenicol are broad-spectrum antibiotics. Although the bacterial resistance mechanisms to these antibiotics have been well documented, hydrolysis of these antibiotics has not been reported in detail. This study reports the hydrolysis of these two antibiotics by a specific hydrolase that is encoded by a gene identified from a soil metagenome. Hydrolysis of chloramphenicol has been recognized in cell extracts of Escherichia coli expressing a chloramphenicol acetate esterase gene, estDL136. A hydrolysate of chloramphenicol was identified as p-nitrophenylserinol by liquid chromatography-mass spectroscopy and proton nuclear magnetic resonance spectroscopy. The hydrolysis of these antibiotics suggested a promiscuous amidase activity of EstDL136. When estDL136 was expressed in E. coli, EstDL136 conferred resistance to both chloramphenicol and florfenicol on E. coli, due to their inactivation. In addition, E. coli carrying estDL136 deactivated florfenicol faster than it deactivated chloramphenicol, suggesting that EstDL136 hydrolyzes florfenicol more efficiently than it hydrolyzes chloramphenicol. The nucleotide sequences flanking estDL136 encode proteins such as amidohydrolase, dehydrogenase/reductase, major facilitator transporter, esterase, and oxidase. The most closely related genes are found in the bacterial family Sphingomonadaceae, which contains many bioremediation-related strains. Whether the gene cluster with estDL136 in E. coli is involved in further chloramphenicol degradation was not clear in this study. While acetyltransferases for chloramphenicol resistance and drug exporters for chloramphenicol or florfenicol resistance are often detected in numerous microbes, this is the first report of enzymatic hydrolysis of florfenicol resulting in inactivation of the antibiotic.

Fig 1

Degradation of chloramphenicol (Cm) by the cell extract of Escherichia coli expressing estDL136. Lane 1, Cm standard; lane 2, Cm (80 μg/ml) incubated with the cell extract of E. coli DH5α carrying pUC119; lanes 3 and 4, crude extracts of E. coli DH5α carrying pUEst136 incubated without and with Cm, respectively. For Cm incubated with cell lysate, reactions were terminated by using the extraction method with butanol. Butanol extracts were concentrated and used for thin-layer chromatography analysis with a developing solvent of methanol/chloroform (1:3). R_f value represents retardation factor, which is the fraction of an analyte in the mobile phase in chromatography.

Fig 2

Identification of the chloramphenicol (Cm) hydrolysate. (A) Comparative analysis of the liquid chromatography-mass spectroscopy profile of purified Cm hydrolysate and p-nitrophenylserinol standard. (B and C) Proton nuclear magnetic resonance (¹H-NMR) analysis of the Cm hydrolysate (B) and p-nitrophenylserinol standard (C).

Fig 3

High-performance liquid chromatography (HPLC) analysis of the hydrolysates of chloramphenicol (Cm) and florfenicol (Ff) by purified EstDL136. (A) Detection of Cm hydrolysis. Peaks 1 and 2 indicate p-nitrophenylserinol and Cm, respectively. (B) Hydrolysis of Ff was achieved following incubation with EstDL136. Peaks 3 and 4 represent the Ff hydrolysate and Ff standard, respectively. mAU, milli-absorbance unit.

Fig 4

Growth of Escherichia coli carrying pUEst136 with different concentrations of chloramphenicol (Cm) or florfenicol (Ff). (A) Bacterial growth during culture with Cm (0 to 128 μg/ml). (B) Bacterial growth during culture with Ff (0 to 128 μg/ml). The dashed line indicates the negative control of E. coli carrying pUC119 cultured with 16 μg/ml of Cm or Ff. OD₆₀₀, optical density at 600 nm.

Fig 5

Genetic organization of ORFs located close to estDL136, and G+C content analysis. Putative ORFs are listed from 1 to 14, and orf8 is estDL136. The G+C content (percentage) of the DNA sequence was calculated using the Seqool program with a 100-bp window; the average G+C content value is estimated to be 53.4%. A DNA scale is also shown (top left).