Corinna Kehrenberg

The Cfr rRNA Methyltransferase Confers Resistance to Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A Antibiotics

ABSTRACT 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 PhLOPSA 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 PhLOPSA 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.

A new mechanism for chloramphenicol, florfenicol and clindamycin resistance: methylation of 23S ribosomal RNA at A2503

The gene product of cfr from Staphylococcus sciuri confers resistance to chloramphenicol, florfenicol and clindamycin in Staphylococcus spp. and Escherichia coli. Cfr is not similar to any other known chloramphenicol resistance determinant. Comparative investigation of E. coli with and without a plasmid‐encoded Cfr showed a decreased drug binding to ribosomes in the presence of Cfr. As chloramphenicol/florfenicol and clindamycin have partly overlapping drug binding sites on the ribosome, the most likely explanation is that Cfr modifies the RNA in the drug binding site. This hypothesis was supported by drug footprinting data that showed both a decreased drug binding and an enhanced reverse transcriptase stop at position 2504, which corresponds to a modification at position A2503 at the drug binding site. A 45 n long RNA fragment containing the appropriate region was isolated and MALDI‐TOF mass spectrometry in combination with tandem mass spectrometry showed an additional methylation at position A2503. Moreover, reduced methylation was detected at nucleotide C2498. The results show that Cfr is an RNA methyltransferase that targets nucleotide A2503 and inhibits ribose methylation at nucleotide C2498, thereby causing resistance to chloramphenicol, florfenicol and clindamycin.

Distribution of florfenicol resistance genes fexA and cfr among chloramphenicol-resistant Staphylococcus isolates.

ABSTRACT 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.

Identification of a Plasmid-Borne Chloramphenicol-Florfenicol Resistance Gene in Staphylococcus sciuri

ABSTRACT The 16.5-kbp plasmid pSCFS1 from Staphylococcus sciurimediated 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.

Diversity of tetracycline resistance genes in bacteria from Chilean salmon farms.

ABSTRACT Twenty-five distinct tetracycline-resistant gram-negative bacteria recovered from four Chilean fish farms with no history of recent antibiotic use were examined for the presence of tetracycline resistance (tet) genes. Sixty percent of the isolates carried 1 of the 22 known tet genes examined. The distribution was as follows. The tet(A) gene was found in six isolates. The tet(B) gene was found in two isolates, including the first description in the genus Brevundimonas. Two isolates carried the tet(34) and tet(B) genes, including the first description of the tet(34) gene in Pseudomonas and Serratia and the first description of the tet(B) gene in Pseudomonas. The tet(H) gene was found in two isolates, which includes the first description in the genera Moraxella and Acinetobacter. One isolate carried tet(E), and one isolate carried tet(35), the first description of the gene in the genus Stenotrophomonas. Finally, one isolate carried tet(L), found for the first time in the genus Morganella. By DNA sequence analysis, the two tet(H) genes were indistinguishable from the previously sequenced tet(H) gene from Tn5706 found in Pasteurella multocida. The Acinetobacter radioresistens isolate also harbored the Tn5706-associated 1,063-bp IS element IS1597, while the Moraxella isolate carried a 1,026-bp IS-like element whose 293-amino-acid transposase protein exhibited 69% identity and 84% similarity to the transposase protein of IS1597, suggesting the presence of a novel IS element. The distribution of tet genes from the Chilean freshwater ponds was different than those that have previously been described from other geographical locations, with 40% of the isolates carrying unidentified tetracycline resistance genes.

Methicillin-resistant and -susceptible Staphylococcus aureus strains of clonal lineages ST398 and ST9 from swine carry the multidrug resistance gene cfr.

ABSTRACT Methicillin-resistant Staphylococcus aureus clonal lineage ST398 and methicillin-susceptible lineage ST9 strains have their main reservoir in swine but can colonize and cause infections in humans. The phenicol/lincosamide/oxazolidinone/pleuromutilin/streptogramin A multidrug resistance gene cfr was detected in isolates of both clonal lineages, rendering a spread to humans with exposure to swine farming possible.

IS21-558 Insertion Sequences Are Involved in the Mobility of the Multiresistance Gene cfr

ABSTRACT During a study of florfenicol-resistant porcine staphylococci from Denmark, the genes cfr and fexA were detected in the chromosomal DNA or on plasmids of Staphylococcus hyicus, Staphylococcus warneri, and Staphylococcus simulans. A novel variant of the phenicol resistance transposon Tn558 was detected on the ca. 43-kb plasmid pSCFS6 in S. warneri and S. simulans isolates. Sequence analysis of a 22,010-bp segment revealed that the new Tn558 variant harbored an additional resistance gene region integrated into the tnpC reading frame. This resistance gene region consisted of the clindamycin exporter gene lsa(B) and the gene cfr for combined resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A antibiotics bracketed by IS21-558 insertion sequences orientated in the same direction. A 6-bp target site duplication was detected at the integration site within tnpC. Transpositionally active forms of the IS21-558 element, known as minicircles, were detected by PCR and suggest that this insertion sequence is involved in the mobility of the multiresistance gene cfr. Based on the knowledge of the transposition pathways of IS21-like insertion sequences and the sequence features detected, the resistance gene region of plasmid pSCFS6 is believed to have developed via IS21-558-mediated cointegrate formation. The data obtained in this study identified the multiresistance gene cfr not only in three novel host species but also in a novel genetic context whose further analysis suggested that insertion sequences of the type IS21-558 are likely to be involved in the dissemination of cfr.

Plasmid-Mediated Florfenicol Resistance Encoded by the floR Gene in Escherichia coli Isolated from Cattle

ABSTRACT A florfenicol resistance gene almost identical to floRof Salmonella enterica serovar Typhimurium DT104 was detected on 110- to 125-kb plasmids in Escherichia coliisolates of animal origin. Analysis of the floR gene flanking regions of one of the plasmids showed that they were different from those encountered in S. enterica serovar Typhimurium DT104.

fexA, a Novel Staphylococcus lentus Gene Encoding Resistance to Florfenicol and Chloramphenicol

ABSTRACT The Staphylococcus lentus plasmid pSCFS2 carries a novel florfenicol-chloramphenicol resistance gene, designated fexA, encoding a protein of 475 amino acids with 14 transmembrane domains. The FexA protein differs from all previously known proteins involved in the efflux of chloramphenicol and florfenicol. Induction of fexA expression by chloramphenicol and florfenicol occurs via translational attenuation.

Decreased fluoroquinolone susceptibility in mutants of Salmonella serovars other than Typhimurium: detection of novel mutations involved in modulated expression of ramA and soxS

OBJECTIVES Mutants of five Salmonella enterica serovars were investigated for structural changes in regulatory regions known to be involved in the up-regulation of efflux pumps. METHODS Five Salmonella field isolates and mutants, in which efflux pump inhibitor tests previously pointed towards an up-regulation of efflux, plus one negative control were included in the study. MIC values were determined of antibiotics that were indicative of AcrAB overexpression. The regulatory regions acrRA, soxRS, marORAB, acrSE and ramRA of original strains and mutants were sequenced and compared. The gene expression of acrA, tolC, ramA and soxS was assessed by quantitative real-time PCR. Conjugation experiments and tet gene PCRs were performed to explain unexpected variations in MIC values of tetracycline. RESULTS In four mutant strains, changes in the ramRA regulatory region, causing up-regulation of ramA, were detected. These changes comprised point mutations and deletions of 10 or 15 bp within the ramR gene and a single bp exchange located in the binding site of the RamR protein in Salmonella Infantis, Paratyphi and Livingstone mutants. An insertion of 49 bp within the soxR gene was involved in soxS up-regulation and enhanced efflux activity in the fifth mutant from Salmonella Virchow. The loss of tetracycline resistance in one Salmonella Paratyphi mutant could be explained by the loss of a plasmid carrying a tet(A) gene. CONCLUSIONS Changes in the ramR-ramA region as well as in the soxR gene occur in mutants of Salmonella serovars other than Typhimurium and seem to be involved in the up-regulation of efflux activity.