Salmonella enterica isolates (n ‫؍‬ 182) were examined for mutations in the quinolone resistance-determining region of gyrA, gyrB, parC, and parE. The frequency, location, and type of GyrA substitution varied with the serovar. Mutations were found in parC that encoded Thr57-Ser, Thr66-Ile, and Ser80-Arg substitutions. Mutations in the gyrB quinolone resistance-determining region were located at codon Tyr420-Cys or Arg437-Leu. Novel mutations were also found in parE encoding Glu453-Gly, His461-Tyr, Ala498-Thr, Val512-Gly, and Ser518-Cys. Although it is counterintuitive, isolates with a mutation in both gyrA and parC were more susceptible to ciprofloxacin than were isolates with a mutation in gyrA alone.The four most common nontyphoidal Salmonella enterica serovars isolated from humans in England and Wales in 1999 were Enteritidis (57%), Typhimurium (18%), Virchow, and Hadar (14). In the last decade, the numbers of isolates of these serovars with decreased susceptibility to ciprofloxacin (MIC, Ն1 g/ml) have increased (12). In 2000, 52% of the S. enterica serovar Virchow isolates tested exhibited decreased susceptibility to ciprofloxacin (12). It is postulated that many of these isolates have arisen in animals after fluoroquinolone exposure and have been transferred to humans via the food chain (11).In salmonellae, where DNA gyrase is the primary target of quinolone action, a single point mutation in the quinolone resistance-determining region (QRDR) of gyrA can mediate resistance to the nonfluorinated quinolone nalidixic acid and reduced susceptibility to fluoroquinolones such as ciprofloxacin, e.g., an MIC of 0.25 g/ml (10). Mutations in the gyrB and topoisomerase IV genes parC and parE are considered rare in salmonellae (6,7,9,11). It was hypothesized that those isolates with decreased susceptibility harbored a single mutation in gyrA, whereas resistant isolates would contain two or more mutations in gyrA and/or gyrB and/or parC and/or parE.S. enterica isolates (n ϭ 182; 156 from animals, 18 from humans, and 18 from the environment) representative of the 25 serotypes typically isolated between 1997 and 2000 were obtained from the Enteric Bacteria Reference Laboratory of the Veterinary Laboratories Agency. Because of the recent debate about the breakpoint concentration of ciprofloxacin (1,8), isolates resistant to Ն0.12 g of ciprofloxacin per ml were investigated. Isolates were serotyped by a microagglutination method (13) and, where appropriate, were phage typed (15). All isolates were from geographically and temporally distinct samples. Isolates were also analyzed by pulsed-field gel electrophoresis, which confirmed that no clones were examined.

The Escherichia coli mob locus is required for synthesis of active molybdenum cofactor, molybdopterin guanine dinucleotide. The mobB gene is not essential for molybdenum cofactor biosynthesis because a deletion of both mob genes can be fully complemented by just mobA. Inactive nitrate reductase, purified from a mob strain, can be activated in vitro by incubation with protein FA (the mobA gene product), GTP, MgCl2, and a further protein fraction, factor X. Factor X activity is present in strains that lack MobB, indicating that it is not an essential component of factor X, but over-expression of MobB increases the level of factor X. MobB, therefore, can participate in nitrate reductase activation. The narJ protein is not a component of mature nitrate reductase but narJ mutants cannot express active nitrate reductase A. Extracts from narJ strains are unable to support the in vitro activation of purified mob nitrate reductase: they lack factor X activity. Although the mob gene products are necessary for the biosynthesis of all E. coli molybdoenzymes as a result of their requirement for molybdopterin guanine dinucleotide, NarJ action is specific for nitrate reductase A. The inactive nitrate reductase A derivative in a narJ strain can be activated in vitro following incubation with cell extracts containing the narJ protein. NarJ acts to activate nitrate reductase after molybdenum cofactor biosynthesis is complete.

Comparative reverse transcription-PCR in combination with denaturing high-pressure liquid chromatography analysis was used to determine the levels of expression of soxS, marA, acrF, acrB, and acrD in multipleantibiotic-resistant (MAR) Salmonella enterica serovar Typhimurium isolates and mutants of S. enterica serovar Typhimurium SL1344 with defined deletions. Posttherapy MAR clinical isolates had increased levels of expression of all genes except soxS. S. enterica serovar Typhimurium SL1344 ⌬acrB expressed 7.9-fold more acrF than the parent strain. A strain with an acrF deletion expressed 4.6-fold more acrB. Deletion of acrB and/or acrF resulted in 2.7-to 4.3-fold more marA mRNA and 3.6-to 4.9-fold increases in the levels of expression of acrD but had a variable effect on the expression of soxS. All mutants were hypersusceptible to antibiotics, dyes, and detergents; but the MIC changes were more noticeable for SL1344 with the acrB deletion than for the mutant with the acrF disruption. These mutants had different but overlapping phenotypes, and the concentrations of ciprofloxacin accumulated by the mutants were different. These data suggest that acrB, acrF, and acrD are coordinately regulated and that their expression influences the expression of the transcriptional activators marA and soxS.

SummaryCytochrome c 552 is the terminal component of the formate-dependent nitrite reduction pathway of Escherichia coli. In addition to four 'typical' haem-binding motifs, CXXCH-, characteristic of c-type cytochromes, the N-terminal region of NrfA includes a motif, CWSCK. Peptides generated by digesting the cytochrome from wild-type bacteria with cyanogen bromide followed by trypsin were analysed by on-line HPLC MS/MS in parent scanning mode. A strong signal at mass 619, corresponding to haem, was generated by fragmentation of a peptide of mass 1312 that included the sequence CWSCK. Neither this signal nor the haemcontaining peptide of mass 1312 was detected in parallel experiments with cytochrome that had been purified from a transformant unable to synthesize NrfE, NrfF and NrfG: this is consistent with our previous report that NrfE and NrfG (but not NrfF) are essential for formate-dependent nitrite reduction. Redox titrations clearly revealed the presence of high and low midpoint potential redox centres. The best fit to the experimental data is for three n ¼ 1 components with midpoint redox potentials (pH 7.0) of þ45 mV (21% of the total absorbance change), ¹90 mV (36% of the total) and ¹210 mV (43% of the total). Plasmids in which the lysine codon of the cysteine-lysine motif, AAA, was changed to the histidine codon CAT (to create a fifth 'typical' haem c-binding motif), or to the isoleucine and leucine codons, ATT and CTT, were unable to transform a Nrf ¹ deletion mutant to Nrf þ or to restore formate-dependent nitrite reduction to the transformants. The presence of a 50 kDa periplasmic c-type cytochrome was confirmed by staining proteins separated by SDS-PAGE for covalently bound haem, but the methyl-viologen-dependent nitrite reductase activities associated with the mutated proteins, although still detectable, were far lower than that of the native protein. The combined data establish not only that there is a haem group bound covalently to the cysteine-lysine motif of cytochrome c 552 but also that one or more products of the last three genes of the nrf operon are essential for the haem ligation to this motif.

Denaturing high-performance liquid chromatography (DHPLC) was evaluated as a rapid screening and identification method for DNA sequence variation detection in the quinolone resistance-determining region of gyrA from Salmonella serovars. A total of 203 isolates of Salmonella were screened using this method. DHPLC analysis of 14 isolates representing each type of novel or multiple mutations and the wild type were compared with LightCycler-based PCR-gyrA hybridization mutation assay (GAMA) and single-strand conformational polymorphism (SSCP) analyses. The 14 isolates gave seven different SSCP patterns, and LightCycler detected four different mutations. DHPLC detected 11 DNA sequence variants at eight different codons, including those detected by LightCycler or SSCP. One of these mutations was silent. Five isolates contained multiple mutations, and four of these could be distinguished from the composite sequence variants by their DHPLC profile. Seven novel mutations were identified at five different loci not previously described in quinolone-resistant salmonella. DHPLC analysis proved advantageous for the detection of novel and multiple mutations. DHPLC also provides a rapid, high-throughput alternative to LightCycler and SSCP for screening frequently occurring mutations.