Inactivation of Chloramphenicol and Florfenicol by a Novel Chloramphenicol Hydrolase

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>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<jats:named-content content-type="genus-species">Escherichia coli</jats:named-content>expressing a chloramphenicol acetate esterase gene,<jats:italic>estDL136</jats:italic>. A hydrolysate of chloramphenicol was identified as<jats:italic>p</jats:italic>-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<jats:italic>estDL136</jats:italic>was expressed in<jats:named-content content-type="genus-species">E. coli</jats:named-content>, EstDL136 conferred resistance to both chloramphenicol and florfenicol on<jats:named-content content-type="genus-species">E. coli</jats:named-content>, due to their inactivation. In addition,<jats:named-content content-type="genus-species">E. coli</jats:named-content>carrying<jats:italic>estDL136</jats:italic>deactivated florfenicol faster than it deactivated chloramphenicol, suggesting that EstDL136 hydrolyzes florfenicol more efficiently than it hydrolyzes chloramphenicol. The nucleotide sequences flanking<jats:italic>estDL136</jats:italic>encode proteins such as amidohydrolase, dehydrogenase/reductase, major facilitator transporter, esterase, and oxidase. The most closely related genes are found in the bacterial family<jats:named-content content-type="genus-species">Sphingomonadaceae</jats:named-content>, which contains many bioremediation-related strains. Whether the gene cluster with<jats:italic>estDL136</jats:italic>in<jats:named-content content-type="genus-species">E. coli</jats:named-content>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.</jats:p>

Tetracycline and Phenicol Resistance Genes and Mechanisms: Importance for Agriculture, the Environment, and Humans

“…These genes code for the aforementioned rRNA methylases and mediate phenicol resistance by target site modification (Kehrenberg et al, 2005: Long et al, 2006Hansen and Vester, 2015). Moreover, a single gene of unknown origin that confers chloramphenicol resistance by hydrolysis has been described (Tao et al, 2012). During whole genome sequencing, cat-like genes have been annotated in the genomes of several bacteria, e.g., Brucella melitensis (GenBank NC_003317) and Bacillus cereus (GenBank NC_004722).…”

Antimicrobial resistance and antimicrobial resistance genes in marine bacteria from salmon aquaculture and non‐aquaculture sites

“…We have also observed that after a few passages in solid and liquid media some of these strains often appear to lose their qnr determinants (A. Tomova and F. C. Cabello, unpublished). Elimination of strains with larger IZD for florfenicol may have similarly biased against strains containing only floR resistance genes and lacking other florfenicol resistance genes (Tao et al, 2012). A large proportion of the AR bacteria from the aquaculture and the non-aquaculture sites did not contain most of the tested genes (Table 1).…”

Novel resistance functions uncovered using functional metagenomic investigations of resistance reservoirs

“…The majority of acquired antibiotic resistance genes in bacterial pathogens are obtained via horizontal gene transfer (HGT) (Ochman et al, 2000), likely with environmental origins (Benveniste and Davies, 1973; Wright, 2010; D'Costa et al, 2011). Accordingly, an increasing impetus has been placed on cataloging antibiotic resistance reservoirs (Allen et al, 2009b, 2010), determining how resistance genes are most readily transferred to the clinic (Marshall and Levy, 2011; Smillie et al, 2011), and identifying resistance mechanisms heretofore unseen in clinical settings (Jeon et al, 2011; Tao et al, 2012). …”

“…The protein changes that enable it to also hydrolyze β-lactams are currently unidentified. A novel chloramphenicol acetate esterase, EstDL136, isolated from a soil metagenome, hydrolyzes the amide linkage of both chloramphenicol and its synthetic derivative florfenicol (Tao, 2011; Tao et al, 2012). Many of the surrounding genes in the metagenomic fragment are most closely related to Sphingomonadaceae, which are frequently considered for bioremediation for their ability to degrade many compounds (Stolz, 2009).…”