Chloramphenicol (Cm) and its fluorinated derivative florfenicol (Ff) represent highly potent inhibitors of bacterial protein biosynthesis. As a consequence of the use of Cm in human and veterinary medicine, bacterial pathogens of various species and genera have developed and/or acquired Cm resistance. Ff is solely used in veterinary medicine and has been introduced into clinical use in the mid-1990s. Of the Cm resistance genes known to date, only a small number also mediates resistance to Ff. In this review, we present an overview of the different mechanisms responsible for resistance to Cm and Ff with particular focus on the two different types of chloramphenicol acetyltransferases (CATs), specific exporters and multidrug transporters. Phylogenetic trees of the different CAT proteins and exporter proteins were constructed on the basis of a multisequence alignment. Moreover, information is provided on the mobile genetic elements carrying Cm or Cm/Ff resistance genes to provide a basis for the understanding of the distribution and the spread of Cm resistance--even in the absence of a selective pressure imposed by the use of Cm or Ff.

A novel multidrug resistance phenotype mediated by the Cfr rRNA methyltransferase is observed in Staphylococcus aureus and Escherichia coli. The cfr gene has previously been identified as a phenicol and lincosamide resistance gene on plasmids isolated from Staphylococcus spp. of animal origin and recently shown to encode a methyltransferase that modifies 23S rRNA at A2503. Antimicrobial susceptibility testing shows that S. aureus and E. coli strains expressing the cfr gene exhibit elevated MICs to a number of chemically unrelated drugs. The phenotype is named PhLOPS A for resistance to the following drug classes: Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A antibiotics. Each of these five drug classes contains important antimicrobial agents that are currently used in human and/or veterinary medicine. We find that binding of the PhLOPS A drugs, which bind to overlapping sites at the peptidyl transferase center that abut nucleotide A2503, is perturbed upon Cfr-mediated methylation. Decreased drug binding to Cfr-methylated ribosomes has been confirmed by footprinting analysis. No other rRNA methyltransferase is known to confer resistance to five chemically distinct classes of antimicrobials. In addition, the findings described in this study represent the first report of a gene conferring transferable resistance to pleuromutilins and oxazolidinones.The bacterial ribosome is the site of protein synthesis and the target for many chemically diverse classes of antimicrobial agents. The antimicrobial drugs target important functional centers of the ribosome and most often bind to rRNA. Recently, a new phenicol and clindamycin resistance phenotype was found to be caused by an RNA methyltransferase designated Cfr. A detailed analysis by drug footprinting studies and matrix-assisted laser desorption-ionization time of flight/tandem mass spectrometry showed that Cfr adds an additional methyl group at position A2503 of 23S rRNA (9). Since A2503 is located in close proximity to the overlapping ribosomal binding sites of phenicols and clindamycin, it was concluded that the Cfr-mediated methylation confers resistance to these two classes of antimicrobial agents by interfering with the positioning of the drugs (9).The cfr gene was first discovered in 2000 during a surveillance study for florfenicol resistance among staphylococci from animals. It was initially detected on the 17.1-kb multiresistance plasmid pSCFS1 from a bovine strain of Staphylococcus sciuri (24) and has also been found in bovine strains of Staphylococcus simulans (6). In addition to cfr, the pSCFS1 plasmid carries the rRNA methylase gene erm(33), the aminocyclitol phosphotransferase gene spc, and the ABC transporter gene lsa(B), which confer resistance to macrolide-lincosamide-streptogramin B (MLS B ) antibiotics, spectinomycin, and lincosamides, respectively. The cfr gene was recently detected on the 35.7-kb plasmid, pSCFS3, from a porcine Staphylococcus aureus strain, together with the chloramphenicol/florfenicol exporter gene f...

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A total of 302 chloramphenicol-resistant Staphylococcus isolates were screened for the presence of the florfenicol/chloramphenicol resistance genes fexA and cfr and their localization on mobile genetic elements. Of the 114 isolates from humans, only a single Staphylococcus aureus isolate showed an elevated MIC to florfenicol, but did not carry either of the known resistance genes, cfr or fexA. In contrast, 11 of the 188 staphylococci from animal sources were considered florfenicol resistant and carried either cfr (one isolate), fexA (five isolates), or both resistance genes (five isolates). In nine cases we confirmed that these genes were carried on a plasmid. Five different types of plasmids could be differentiated on the basis of their sizes, restriction patterns, and resistance genes. The gene fexA, which has previously been shown to be part of the nonconjugative transposon Tn558, was identified in 10 of the 11 resistant isolates from animals. PCR assays were developed to detect different parts of this transposon as well as their physical linkage. Complete copies of Tn558 were found in five different isolates and shown by inverse PCR to be functionally active. Truncated copies of Tn558, in which the tnpA-tnpB area was in part deleted by the integration of a 4,674-bp segment including the gene cfr and a novel 2,446-bp IS21-like insertion sequence, were seen in a plasmid present in three staphylococcal isolates.

The 16.5-kbp plasmid pSCFS1 from Staphylococcus sciuri mediated combined resistance to chloramphenicol and florfenicol. The gene responsible for this resistance property, cfr, was cloned and sequenced. The amino acid sequence of the Cfr protein revealed no homology to known acetyltransferases or efflux proteins involved in chloramphenicol and/or florfenicol resistance or to other proteins whose functions are known.Staphylococcus sciuri is a common inhabitant of the physiological skin flora of most rodents, ungulates, carnivora, and marsupials. Although classified as rarely pathogenic (6), S. sciuri isolates have been obtained occasionally from cases of mastitis in goats (10) and bronchopneumonia in cattle (13). Antimicrobial resistance is common among S. sciuri isolates, and a number of plasmids carrying one or more resistance genes have been identified (11,13,14). Resistance to chloramphenicol (CM) in staphylococci has usually been associated with plasmid-borne cat genes (11, 13), whose gene products inactivate CM by diacetylation. CM acetyltransferases, however, are unable to inactivate florfenicol (FF), a fluorinated CM derivative which was licensed in Germany in 1995 as a therapeutic agent to control bacterial respiratory infections in cattle. Genes whose gene products mediate combined resistance to CM and FF by efflux of both drugs have been identified in gram-negative bacteria, such as Salmonella enterica serovar Typhimurium (2) and Photobacterium damselae subsp. piscicida, formerly known as Pasteurella piscicidae (5). In staphylococci and related organisms, FF resistance genes have not been described yet.An S. sciuri isolate obtained from the nasal swab of a calf suffering from an infection of the respiratory tract proved to be resistant to tetracycline, erythromycin, kanamycin, CM, and FF. Plasmid analysis revealed the presence of six plasmids in the size range between 1.5 and 16.5 kbp. Experiments involving transformation into protoplasts of Staphylococcus aureus RN4220 (12) and subsequent selection of the transformants on regeneration media containing 20 g of FF/ml (Essex, Munich, Germany) identified only the 16.5-kbp plasmid, designated pSCFS1, as the mediator of resistance to CM and FF. This plasmid also mediated resistance to erythromycin by an inducibly expressed ermC gene as confirmed by PCR analysis (7). Cloning experiments revealed that the ermC gene was located on a 2.5-kbp PstI fragment of pSCFS1 (data not shown). The original S. sciuri isolate and S. aureus RN4220:pSCFS1 showed FF MICs of 64 g/ml and CM MICs of 32 g/ml. Preincubation of these isolates in the presence of either 0.5 g of FF or 0.5 g of CM increased the FF MICs to 512 g/ml and the CM MICs to 64 g/ml, suggesting that pSCSF1-mediated resistance to FF and CM in both staphylococcal hosts is inducible by FF as well as CM. Plasmid pSCFS1 was mapped ( Fig. 1) and subjected to cloning experiments. Restriction fragments of pSCFS1 generated by the enzymes EcoRI and BclI-BamHI were cloned into pBluescript SKII ϩ . The recombinant plasmids ...