Thermotolerant Campylobacter species C. jejuni and C. coli are actually recognized as the major bacterial agent responsible for food-transmitted gastroenteritis. The most effective antimicrobials against Campylobacter are macrolides and some, but not all aminoglycosides. Among these, susceptibility to streptomycin is reduced by mutations in the ribosomal RPSL protein or by expression of ANT(6)-I aminoglycoside O-nucleotidyltransferases. The presence of streptomycin resistance genes was evaluated among streptomycin-resistant Campylobacter isolated from humans and animals by using PCR with degenerated primers devised to distinguish ant(6)-Ia, ant(6)-Ib and other ant-like genes. Genes encoding ANT(6)-I enzymes were found in all possible combinations with a major fraction of the isolates carrying a previously described ant-like gene, distantly related and belonging to the new ant(6)-I sub-family ant(6)-Ie. Among Campylobacter isolates, ant(6)-Ie was uniquely found functional in C. coli, as shown by gene transfer and phenotype expression in Escherichia coli, unlike detected coding sequences in C. jejuni that were truncated by an internal frame shift associated to RPSL mutations in streptomycin resistant strains. The genetic relationships of C. coli isolates with ANT(6)-Ie revealed one cluster of strains presented in bovine and humans, suggesting a circulation pathway of Campylobacter strains by consuming contaminated calf meat by bacteria expressing this streptomycin resistance element.

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Campylobacter is one of the most important microorganisms responsible for foodborne diseases in the EU. In this study, we investigated resistance to tetracycline in 139 Campylobacter jejuni and Campylobacter coli samples isolated from human clinical cases. From these, 110 were resistant to tetracycline, with MIC (minimal inhibitory concentration) varying in a range of 1 to >512 μg/mL, and 109 (78.4%) carried tet(O), a gene that confers resistance to tetracycline through the expression of a protein that confers protection to the ribosome. Amongst the tetracycline-resistant isolates, one C. jejuni (HCC30) was the only tet(O)-negative sample, presenting an MIC of 256 μg/mL. Instead, the mosaic gene tet(O/M/O) was found in HCC30 and, as far as we know, this is the first description of this chimeric gene originating from homologous recombination between tet(O) and tet(M). The previously described mosaic gene tet(O/32/O), also found in Campylobacter, presents a chimeric structure very similar to that of tet(O/M/O), affecting domains II and III of encoded proteins distantly related to the elongation factor G (EF-G). The tet(O/M/O) mosaic gene has been found in nucleotide databases in several genomes of Campylobacter isolated from different origins, indicating its frequent acquisition, even though it can be undetected through screening by PCR with specific tet(O) primers. In this work, we address the improvement of classical PCR to efficiently diagnose the most prevalent tetracycline resistance determinants in Campylobacter, including tet(O/M/O), which should be taken into account in the optimization of campylobacteriosis treatments.

A screening of antimicrobial resistance and its genetic determinants has been performed on 300 Salmonella enterica isolates collected during 2004-2008 from human infections in Spain. Salmonella Typhimurium and Salmonella Enteritidis were the major serotypes, which were found with similar frequencies covering 80% of the bacterial collection. Salmonella Typhimurium isolates frequently shared low susceptibility to antimicrobials of the penta-resistance phenotype (ACSSuT) and/or cephalosporin resistance. The ACSSuT profile was found closely linked to int1-associated gene cassettes, with major elements carrying DNA fragments of 1.0 Kb (aadA2 gene) plus 1.2 Kb (blaPSE-1 gene) or 2.0 Kb (aadA1 and blaOXA-1 genes). Among these, ACSSuT and cephalosporin resistances were associated in Salmonella Typhimurium isolates expressing the blaOXA gene. β-lactamase activities were also detected from isolates carrying blaTEM, blaCMY, or blaSHV, although only the two last genes expressed extended-spectrum β-lactamases. The clonal analysis of S. enterica strains suggests that both horizontal and vertical transfer mechanisms are involved in the wide dissemination of their antimicrobial resistance.

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...