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We identified a quinolone resistance locus,flqB, linked to transposon insertion Q1108 andfjis on the SmaI D fragment of the Staphylococcus aureus NCTC 8325 chromosome, the same fragment that contains the norA gene. S. aureus norA cloned fromflqB andflqB+ strains in Escherichia coli differed only in a single nucleotide in the putative promoter region. There was no detectable change in the number of copies of norA on the chromosomes of flqB strains, but they had increased levels of norA transcripts. Cloned norA produced resistance to norfloxacin and other hydrophilic quinolones and reduced norfloxacin accumulation in intact cells that was energy dependent, suggesting active drug efflux as the mechanism of resistance. Drug efflux was studied by measurement of norfloxacin uptake into everted inner membrane vesicles prepared from norA-containing E. coli cells. Vesicles exhibited norfloxacin uptake after the addition of lactate or NADH, and this uptake was abolished by carbonyl cyanide m-chlorophenylhydrazone and nigericin but not valinomycin, indicating that it was linked to the pH gradient across the cell membrane. Norfloxacin uptake into vesicles was also saturable, with an apparent Km of 6 ,zM, a concentration between those that inhibit the growth offlqB and flqB+ S. aureus cells, indicating that drug uptake is mediated by a carrier with a high apparent affinity for norfloxacin. Ciprofloxacin and ofloxacin competitively inhibited norfloxacin uptake into vesicles. Reserpine, which inhibits the multidrug efflux mediated by the bmr gene of Bacilus subtilis, which is similar to norA, abolished norfloxacin uptake into vesicles as well as the norfloxacin resistance of anflqB mutant, suggesting a potential means for circumventing quinolone resistance as a result of drug elux in S. aureus. These fiidings indicate that the chromosomal flqB resistance locus is associated with increased levels of expression of norA and strongly suggest that the NorA protein itself functions as a drug transporter that is coupled to the proton gradient across the cell membrane.Fluoroquinolones such as norfloxacin, ciprofloxacin, and ofloxacin are broad-spectrum, synthetic antimicrobial agents that are widely used for the treatment of bacterial infections. Development of drug resistance in some species, particularly Pseudomonas aeruginosa and Staphylococcus aureus, has, however, limited the utilities of fluoroquinolones in some clinical settings. The genetics and mechanisms of bacterial fluoroquinolone resistance have been studied most extensively in Escherichia coli and P. aeruginosa (9). These drugs act on DNA gyrase, and in E. coli, mutations in the gyrA and gyrB genes encoding the DNA gyrase A and B subunits, respectively, have been shown to cause resistance (6,11,38,40). In addition, mutations in genes that affect the expression of porin outer membrane proteins have been shown to cause resistance and to be associated with reduced levels of drug accumulation in intact cells (10,11). Moreover, in these mutant gram-negative bacteria, reduced ...

Vibrio cholerae causes the potentiafly lethal disase cholera through the elaboration of the intestinal secretogen cholera toxin. A second toxin of V. cholerae, Zot, decreases intestinal tissue resistance by modifying interceflular tight junctions. In this report, a third toxin of V. cholerae, Ace (accessory cholera enterotoxin), is described. Ace increases short-circuit current in Ussing chambers and causes fluid secretion in ligated rabbit ileal loops. The predicted protein sequence of Ace shows striking similarit to eukaryotic iontransporting ATPases, including the product of the cystic fibrosis gene. The gene encoding Ace is located immediately upstream of the genes encoding Zot and cholera toxin. The ctx, zot, and ace genes, which are located on a dynamic sector of the chromosome, comprise a V. cholerae "virulence cassette." Development oflive attenuated Vibrio cholerae oral vaccines by recombinant technology initially targeted the genes encoding cholera toxin (ctx) for deletion. Although these initial vaccine candidates, such as JBK70 and CVD101, elicited high antibody responses and did not cause severe diarrhea in volunteers, more than half ofthe vaccinees developed mild to moderate diarrhea and many experienced abdominal cramps, anorexia, and low-grade fever (1). In search of an additional toxin which could explain this reactogenicity, we reported a second toxin, Zot (zonula occludens toxin), which decreases ileal tissue resistance by affecting intercellular tightjunctions (2). The diarrhea observed in volunteers fed Actx V. cholerae vaccine strains was hypothesized to be caused by alteration of tight junctions, with a subsequent increase in intestinal permeability.The gene encoding Zot is located immediately upstream of ctx (3) on a 4.5-kb region termed the "core region" (4). In many strains of V. cholerae, this 4.5-kb region is flanked by one or more copies of a 2.7-kb sequence called RS1; homologous recombination between RS1 elements can lead to tandem amplification of the 4.5-kb region (4). Since the zot and ctx genes comprise only 55% of the 4.5-kb core region, we investigated the potential pathogenic role ofthe remaining portion of this region. Our results show that the gene for a third toxin is included in this regionll and that this third toxin increases the short-circuit current (IS) in Ussing chambers and causes fluid secretion in ligated rabbit ileal loops. MATERIALS AND METHODSBacterial Strains. V. cholerae E7946 is an El Tor Ogawa strain previously demonstrated to produce cholera in volunteers (5). Strain CVD110 was constructed from E7946 by homologous recombination of the RS1 elements, resulting in deletion of the core region (J.M., J.E.G., A.F., and J.B.K.,The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. unpublished work). In brief, cloned ctx and zot sequences were inserted into the suicide vector pGP704 (7) and conjugated ...

Mutations in the flqA (formerly ofx/cfx) resistance locus of Staphylococcus aureus were previously shown to be common after first-step selections for resistance to ciprofloxacin and ofloxacin and to map on the S. aureus chromosome distinctly from gyrA, gyrB, and norA.grlA and grlB, the genes for the topoisomerase IV of S. aureus, were identified from a genomic lambda library on a common KpnI fragment, and grlB hybridized specifically with the chromosomal SmaI A fragment, which contains the flqA locus. Amplification of grlA sequences (codons 1 to 251) by PCRs from nine independent single-step flqA mutants, one multistep mutant, and the parent strain identified mutations encoding a change from Ser to Phe at position 80 in four mutants, a novel change from Ala to either Glu or Pro at position 116 in three mutants, and no change in three mutants. In the multistep mutant, another resistance locus, flqC, was mapped by transformation to the chromosomal SmaI G fragment by linkage to omega(ch::Tn551)1051 (58%) and nov (97.9%), which encodes resistance to novobiocin. This fragment contains the gyrA gene, and flqC mutants had a mutation in gyrA encoding a change from Ser to Leu at position 84, a change previously found in resistant clinical isolates. In genetic outcrosses, the flqC (gyrA) mutation expressed resistance only in flqA mutants, including those with both types of grla mutations. The silent mutant allele of gyrA was present in a flqA background and expressed resistance only upon introduction of a grlA mutation. At fourfold the MIC of ciprofloxacin, the bactericidal activity of ciprofloxacin was reduced in a grlA mutant and was abolished in gyrA grlA double mutants. These findings provide direct genetic evidence that topoisomerase IV is the primary target of current fluoroquinolones in S. aureus and that this effect may result from the greater sensitivity of topoisomerase IV relative to that of DNA gyrase to these agents. Furthermore, resistance from an altered DNA gyrase requires resistant topoisomerase IV for its expression.