Q uinolone resistance in Enterobacteriaceae is mediated by mutations in the quinolone resistance-determining regions (QRDR) of topoisomerase genes and/or by plasmid-mediated quinolone resistance determinants (PMQR) such as the qnr genes encoding pentapeptide repeat proteins (1). The qnrB family is represented by 80 different alleles (http://www.lahey.org/qnr Studies/); most of them originated from Citrobacter strains and spread to other Enterobacteriaceae species (2). This work describes the identification of a new allele of the quinolone resistance protein QnrB, QnrB54, in a human clinical isolate of Citrobacter freundii detected in Spain. Isolate HLR20 was detected in October 2007, in a fecal sample of a 53-year-old man treated in the Hospital of Llerena. Identification of the isolate as C. freundii was performed by API 20E testing (bioMérieux), by sequencing its 16S rRNA gene (HG974539) and dnaJ gene (3) (LN624598) and by using a Vitek matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system (bioMérieux). The bacterium showed resistance (4) to ciprofloxacin (CIP; MIC, 4 g/ ml) and nalidixic acid (NAL; MIC, Ͼ512 g/ml), and, accordingly, it presented mutations T83I and S80I in the QRDR of GyrA (5) and ParC (6), respectively. In addition, the screening for PMQR determinants (7-12) detected a qnrB gene in a region that was amplified by PCR with primers FWD (5=-CGCGCGGACCT GCTGGATCGTCT-3=) and REV (5=-TTGCGGGTTGAACGTAT TGACCT-3=) (AB734053), producing a 2,409-bp DNA fragment (HE820727). The sequence of qnrB corresponded to a new allele of QnrB, QnrB54, spanning variants G7S-S79A-I142M-A144T-V212I (http://www.lahey.org/qnrStudies/). The sequence downstream of qnrB (Table 1) encodes a protein with the CaMKIIAD signature (cl17504), a linkage that has been observed in Klebsiella plasmids (13) and Citrobacter chromosomes (2, 14). The qnrB54 gene was shown to encode a functional protein by its amplification with primers FWD (5=-CATCAGCTTCGCGCTTTG-3=) and REV (5=-CCCGCTACACATTCACTTATGC-3=) (HE820727) and cloning in pGEM-Teasy vector (Promega) and Escherichia coli J53 cells; the transformants showed MICs for CIP and NAL of 0.25 g/ml and 16 g/ml, respectively. The conjugation potential of qnrB was attempted by mating performed between isolate HLR20 and E. coli J53, but quinolone resistance was not mobilized. In addition, pulsed-field gel electrophoresis (PFGE) (14) of the HLR20 DNA digested by the enzyme I-CeuI and hybridized to a digoxigenin (DIG)-labeled qnrB probe (Roche) produced a signal, among all the bands detected by a 23S rRNA gene probe, matching an I-ceuI band of 450 to 500 kb, a size much larger than that of any known plasmid of Citrobacter. The description of the QnrB54 allele, expressed from the chromosome of C. freundii, might provide information of interest to trace the spread of quinolone resistance determinants between Enterobacteriaceae. Nucleotide sequence accession numbers. The GenBank accession numbers for the sequences determined in this work are HG974539, LN6...