Mutations In Gyrase Research Articles

Fluoroquinolone and Quinazolinedione Activities against Wild-Type and Gyrase Mutant Strains of Mycobacterium smegmatis

Quinazolinediones (diones) are fluoroquinolone-like inhibitors of bacterial gyrase and DNA topoisomerase IV. To assess activity against mycobacteria, C-8-methoxy dione derivatives were compared with cognate fluoroquinolones by using cultured Mycobacterium smegmatis. Diones exhibited higher MIC values than fluoroquinolones; however, MICs for fluoroquinolone-resistant gyrA mutants, normalized to the MIC for wild-type cells, were lower. Addition of a 3-amino group to the 2,4-dione core increased relative activity against mutants, while alteration of the 8-methoxy group to a methyl or of the 2,4-dione core to a 1,3-dione core lowered activity against mutants. A GyrA G89C bacterial variant was strikingly susceptible to most of the diones tested; in contrast, low susceptibility to fluoroquinolones was observed. Many of the bacteriostatic differences between diones and fluoroquinolones were explained by interactions at the N terminus of GyrA helix IV revealed by recently published X-ray structures of drug-topoisomerase-DNA complexes. When lethal activity was normalized to the MIC in order to minimize the effects of drug uptake, efflux, and ternary complex formation, a 3-amino-2,4-dione exhibited killing activity comparable to that of a cognate fluoroquinolone. Surprisingly, the lethal activity of the dione was inhibited less by chloramphenicol than that of the cognate fluoroquinolone. This observation adds the 2,4-dione structural motif to the list of structural features known to impart lethality to fluoroquinolone-like compounds in the absence of protein synthesis, a phenomenon that is not explained by X-ray structures of drug-enzyme-DNA complexes.

Parallel evolution and local differentiation in quinolone resistance in Pseudomonas aeruginosa

The emergence and spread of antibiotic resistance in pathogens is a major impediment to the control of microbial disease. Here, we review mechanisms of quinolone resistance in Pseudomonas aeruginosa, an important nosocomial pathogen and a major cause of morbidity in cystic fibrosis (CF) patients. In this quantitative literature review, we find that mutations in DNA gyrase A, the primary target of quinolones in Gram-negative bacteria, are the most common resistance mutations identified in clinical samples of all origins, in keeping with previous observations. However, the identities of non-gyrase resistance mutations vary systematically between samples isolated from CF patients and those isolated from acute infections. CF-derived strains tend to harbour mutations in the efflux pump regulator nfxB, while non-CF strains tend to bear mutations in the efflux regulator mexR or in parC, which encodes one of two subunits of DNA topoisomerase IV. We suggest that differences in resistance mechanisms between CF and non-CF strains result either from local adaptation to different sites of infection or from differences in mutational processes between different environments. We further discuss the therapeutic implications of local differentiation in resistance mechanisms to a common antibiotic.

Mutations in gyrase and topoisomerase genes associated with fluoroquinolone resistance in Salmonella serovars from retail meats

Mutations in gyrase and topoisomerase genes (gyrA, gyrB, parC and parE) were examined among 30 multidrug-resistant (MDR) Salmonella isolates recovered from retail meats using PCR and DNA sequencing analysis. Six isolates had a single gyrA mutation (Ser83→Phe or Ser83→Tyr or Asp87→Asn) with ciprofloxacin (Cip) minimum inhibitory concentrations (MICs) ranging from 0.125 to 0.5μg/ml except 1 with 16μg/ml. Three isolates had both a gyrA mutation (Ser83→Tyr or Ser83→Phe) and a parC mutation (Ser80→Arg) (Cip MICs, 8 to 16μg/ml). Fifteen isolates had both double mutations in gyrA (Ser83→Phe and Asp87→Gly or Asp87→Asn) and a single mutation (Ser80→Arg) in parC (Cip MICs, 8 to ≥16μg/ml). Three had double gyrA mutations (Ser83→Phe, Asp87→Gly or Asp87→Asn), one parC mutation (Ser80→Arg), and 1 parE mutation (Lys428→Gln or Gly442→Ser) (Cip MICs, 8 to ≥16μg/ml). One had double gyrA mutations (Ser83→Phe, Asp87→Gly or Asp87→Asn), one parC mutation (Ser80→Arg), and 2 parE mutations (Lys441→Ile and Asp494→Asn) (Cip MICs, 8 to ≥16μg/ml). Isolates with single gyrA mutations were less resistant to quinolone and fluoroquinolones than those together with additional parC and/or parE mutations in Salmonella. Our findings confirmed the importance of point mutations in gyrase and topoisomerase in reduced susceptibility to quinolone and fluoroquinolones. Mutations in gyrA confer low-level quinolone and fluoroquinolone resistance, while additional gyrA mutation(s) together with parC and/or parE mutations increase the resistance to a high level.

Contribution of mutations in DNA gyrase and topoisomerase IV genes to ciprofloxacin resistance in Escherichia coli clinical isolates

DNA gyrase (GyrA and GyrB) and topoisomerase IV (ParC and ParE) are the two essential type II topoisomerases in Escherichia coli. These enzymes act via inhibition of DNA replication. Mutations in the quinolone resistance-determining region (QRDR) of the gyrA, gyrB, parC and parE genes from clinical isolates of E. coli were determined by DNA sequencing of 54 ciprofloxacin-resistant clinical isolates from a hospital in Delhi, India. The majority of the E. coli isolates were shown to carry mutations in gyrA, parC and parE. Ciprofloxacin resistance due to accumulation of such a high number of mutations in the QRDR regions of gyrA at positions Ser83 and Asp87 and parC at position Ser80 as well as outside of the QRDR region of parE at Ser458 and Glu460 confers high-level resistance of ciprofloxacin in clinical isolates. The high frequency of occurrence of mutations in the parE gene (44.4% strains) is alarming, as topoisomerase IV is a secondary target of quinolones.

Emergence of resistant salmonella spp.; new challenges, new trends.

Salmonella enterica is a common cause of human food borne gastroenteritis and bacteremia worldwide. Salmonellosis is caused by different serotypes of salmonella; humans and animals are generally infected with these bacteria by the consumption of contaminated food products (1–3). Non-typhi salmonella gastroenteritis in contrast to the invasive salmonellosis that requires prompt antibiotic therapy, is usually self-limited (4). According to the WHO license for the treatment of salmonellosis in last decades, increasing usage of antimicrobial agents in clinics and slaughterhouses is becoming a major problem (5). Chloramphenicol, sulfamethoxazole-trimethoprim, and ampicillin were the earliest antibiotics used the treatment of salmonellosis. However, following the development of resistance to the aforementioned drugs, quinolones and cephalosporines are currently in use (6). The usage of quinolones and cephalosporines in the veterinary world as the therapeutic agents or growth factors led to the emergence of highly resistant veterinary Salmonella strains which might transfer into human via the food chain process. This transmission will cause failure in clinical treatment of related diseases (7, 8). Frequency of antimicrobial resistance found among isolates from human is directly linked to their use in animals (9). Evaluation of the results of regional studies especially in the countries with uncontrolled use of antibiotics has revealed a significant increase in the number of resistant strains, principally in animal foods as growth factors or therapeutic agents (10–12). Resistance to the aforementioned antibiotics is either chromosomal or plasmid-encoded. Different molecular mechanisms are responsible for the occurrence of resistance against antibiotics in Salmonella spp. (13). Transmissibility of the plasmid-encoded resistant phenotypes among enteric bacteria within the gastrointestinal tract and environment has resulted to a change in the use of the first-line antibiotics against salmonellosis towards antibiotics with no identified similar resistant mechanisms, i.e., quinolones, as the last choice in treatment strategy. New studies in this context have demonstrated the emergence of great numbers of quinolone resistant serotypes in recent years (6, 14, 15). Studies about the aforementioned bacteria have revealed new mechanism of resistance for this group of antibiotics. Mutation in DNA gyrase A/B, topoisomerase IV and plasmid-mediated resistance due to (Qnr), Aac (6′)-Ib-cr, and QepA efflux genes in both of human and animal isolates are three major types of resistance mechanisms in these bacteria. Transmission of these plasmids from other enteric bacteria and natural selection of chromosomally mutated strains could be responsible for the emergence of this type of resistance (16). On the basis of our findings, regarding the regional screening study of the resistance patterns among different S. enterica serotypes isolated from humans, the highest rates of antibiotic resistance were as follows: nalidixic acid; 45.7%, trimethoprim-sulfamethoxazole; 36.4%, ampicillin 15.5%, chloramphenicol; 14.7%, and cephalothin; 6.2% (11). Similar result was found in another study in Iran (12). Most of these isolates have carried mutations on gyrA gene (14). In a new study in USA, the plasmid harboring quinolon resistant bacteria was identified in all of the plasmid harboring human-derived non-typhi enterica isolates of Salmonella spp. (17). According to the report of National Antimicrobial Resistance Monitoring System (NARMS), the increase in MIC (≥ 32 µg/ml) for nalidixic acid in non typhoidal salmonella as the last-line antibiotic and increase in their resistance rates from 0.9% to 2% during 1997 – 2008 (for human isolates of Salmonella enterica serovar Enteritidis about 1.7% to 6.6%), the need for alternative therapeutic approaches seems necessary (18). It seems foresighted that prescription of new antibiotics in future will not guarantee the successful treatment as a result of present resistance mechanisms in these bacteria. Eradication of bacteria from their animal sources seems to be the best inhibitory manner for emergence of resistant strains among these bacteria. Nowadays usage of probiotics and prebiotics and competitive exclusion (CE) cultures are considered as successful and acceptable treatments strategies. These methods can help us to control colonization and growth of gastrointestinal pathogens (19). Exploitation of the mucosal immune system against these bacteria is another useful strategy. Vaccination of animals with an inactived salmonella ghost or its related DNA-based vaccines is a suitable method to prevent bacterial colonization in these bacteria. In addition, use of preservative food additives will reduce microbial infections (20, 21). However, the elimination of any possible source of infection for Salmonella spp. instead of antibiotic therapy seems to be a well- advised solution for controlling the emergence of more resistant Salmonella serotypes.

Reduced Levofloxacin Susceptibility in Clinical Respiratory Isolates ofHaemophilus InfluenzaeIs Not yet Associated with Mutations in the DNA Gyrase and Topoisomerase II Genes in Korea

Among 155 clinical respiratory isolates of Haemophilus influenzae in Korea, 6 (3.9%) isolates had reduced levofloxacin susceptibility (MICs ≥ 0.5 µg/mL). These six isolates had no significant quinolone resistance-determining region (QRDR) mutations in gyrA, gyrB, parC, or parE. This phenomenon suggests that neither evolution nor spread of any significant QRDRs mutations in clinical isolates occurred in Korea. Therefore, continued surveillance is necessary to observe the evolution of antibiotic-resistance and take measures to avoid the spread of drug-resistant clones.

Characterization of antimicrobial resistance in Salmonella enterica food and animal isolates from Colombia: identification of a qnrB19-mediated quinolone resistance marker in two novel serovars

Ninety-three Salmonella isolates recovered from commercial foods and exotic animals in Colombia were studied. The serotypes, resistance profiles and where applicable the quinolone resistance genes were determined. Salmonella Anatum (n=14), Uganda (19), Braenderup (10) and Newport (10) were the most prevalent serovars, and resistance to tetracycline (18.3%), ampicillin (17.2%) and nalidixic acid (14%) was most common. Nalidixic acid-resistant isolates displayed minimum inhibitory concentrations ranging from 32 to 1024 μg mL(-1) . A Thr57→Ser substitution in ParC was the most frequent (12 of the 13 isolates). Six isolates possessed an Asp87→Tyr substitution in GyrA. No alterations in GyrA in a further seven nalidixic acid-resistant isolates were observed. Of these, four serovars including two Uganda, one Infantis and a serovar designated 6,7:d:-, all carried qnrB19 genes associated with 2.7 kb plasmids, two of which were completely sequenced. These exhibited 97% (serovar 6,7:d:- isolate) and 100% (serovar Infantis isolate) nucleotide sequence identity with previously identified ColE-like plasmids. This study demonstrates the occurrence of the qnrB19 gene associated with small ColE plasmids hitherto unrecognized in various Salmonella serovars in Colombia. We also report unusual high-level quinolone resistance in the absence of any DNA gyrase mutations in serovars S. Carrau, Muenchen and Uganda.

Should Moxifloxacin Be Used for the Treatment of Extensively Drug-Resistant Tuberculosis? An Answer from a Murine Model

The prevalence of extensively drug-resistant tuberculosis (XDR-TB), defined as TB that is resistant to isoniazid, rifampin, fluoroquinolones, and aminoglycosides, is rising worldwide. The extent of Mycobacterium tuberculosis resistance to fluoroquinolones depends on the mutation in the DNA gyrase, the only target of fluoroquinolones. The MIC of moxifloxacin, the most active fluoroquinolone against M. tuberculosis, may be lower than its peak serum level for some ofloxacin-resistant strains of Mycobacterium tuberculosis. Therefore, if the MIC of moxifloxacin is lower than its peak serum level, it may be effective against XDR-TB. Our objective was to determine the efficacy of moxifloxacin in treating ofloxacin-resistant TB. We selected isogenic fluoroquinolone-resistant mutants of M. tuberculosis H37Rv in vivo. We infected Swiss mice with either wild-type H37Rv or one of three mutant strains with different MICs that are commonly seen in clinical practice. The MICs of the mutant strains ranged from below to above the peak moxifloxacin level seen in humans (3 μg/ml). Each mouse was treated with one of four moxifloxacin doses for 1 month. Moxifloxacin was effective against mutant strain GyrB D500N, with the lowest MIC (0.5 μg/ml), when the standard dose was doubled. Moxifloxacin reduced mortality in mice infected with mutant strain GyrA A90V with an intermediate MIC (2 μg/ml). However, it had no impact on the mutant strain GyrA D94G with the highest MIC (4 μg/ml). Our study underscores current WHO recommendations to use moxifloxacin when there is resistance to early-generation fluoroquinolones such as ofloxacin, restricting this recommendation to strains with moxifloxacin MICs of less than or equal to 2 μg/ml.

Fluoroquinolone Resistance in Campylobacter

Campylobacter is a commensal in poultry, and therefore, poultry and poultry products are major sources of Campylobacter infections in humans. Fluoroquinolones inhibit the growth of Campylobacter and other microorganisms by binding to bacterial DNA gyrase and DNA topoisomerase IV. These enzymes are associated with bacterial transcription, replication, and chromosome condensation and segregation. Selection pressure in the presence of fluoroquinolones rapidly leads to resistance in Campylobacter, due to the selection for mutations in DNA gyrase. Fluoroquinolone-resistant campylobacters have been found in poultry feces and carcasses, and in retail poultry meat products in most areas of the world. In addition, other food animals and the meat products from those animals have been shown contaminated with fluoroquinolone-resistant campylobacters. Even the removal of fluoroquinolones from use in treating animal diseases has not entirely eliminated the presence of resistant Campylobacter jejuni and Campylobacter coli from animals and animal products. Human exposure to Campylobacter infection could be reduced by using strategies that decrease colonization of chickens by the pathogen.

Characterization of Quinolone Resistance in Escherichia coli Strains of Animal Origin from Italy

The aim of this study was to assess the possible circulation of genetic resistance determinants and chromosomal point mutations in quinolone-resistant Escherichia coli isolated from livestock from central italy. forty-nine E. coli isolates were recovered from animals during the surveillance activities of the Istituto Zooprofilattico Abruzzo e Molise (IZSA&M), Italy, over 2 years. the plasmid resistance determinants and point mutations in DNA gyrase and topoisomerase IV were characterized by PCR and DNA sequencing. Of the 49 E. coli isolates, 34 were resistant to nalidixic acid, 4 to ciprofloxacin and 11 to nalidixic acid, ciprofloxacin and enrofloxacin. Chromosomal point mutations were found in gyrA gene (Ser83Leu and Asp87Asn) and gyrB (Gln434His, Lys444Arg and Gly435Val). We also report the simultaneous presence of qnrS1 quinolone resistance determinant dfrA1-aadA22 gene cassettes and amino acid substitution Ser83Leu in the gyrA gene in an E. coli strain resistant only to nalidixic acid.

Fluoroquinolone Resistance andClostridium difficile, Germany

We characterized 670 Clostridium difficile isolates collected from patients in 84 hospitals in Germany in 2008. PCR ribotyping showed high prevalence of ribotype 001 and restricted dissemination of ribotype 027 strains. Fluoroquinolone resistance and associated gyrase mutations were frequent in various ribotypes, but no resistance to metronidazole or vancomycin was noted.

Coordination of genomic structure and transcription by the main bacterial nucleoid‐associated protein HU

The histone-like protein HU is a highly abundant DNA architectural protein that is involved in compacting the DNA of the bacterial nucleoid and in regulating the main DNA transactions, including gene transcription. However, the coordination of the genomic structure and function by HU is poorly understood. Here, we address this question by comparing transcript patterns and spatial distributions of RNA polymerase in Escherichia coli wild-type and hupA/B mutant cells. We demonstrate that, in mutant cells, upregulated genes are preferentially clustered in a large chromosomal domain comprising the ribosomal RNA operons organized on both sides of OriC. Furthermore, we show that, in parallel to this transcription asymmetry, mutant cells are also impaired in forming the transcription foci-spatially confined aggregations of RNA polymerase molecules transcribing strong ribosomal RNA operons. Our data thus implicate HU in coordinating the global genomic structure and function by regulating the spatial distribution of RNA polymerase in the nucleoid.

Comparison of the metabolic activities of four wild-type Clostridium perfringens strains with their gatifloxacin-selected resistant mutants

The production of short-chain fatty acids, reductive enzymes, and hydrolytic enzymes by four gatifloxacin-selected, fluoroquinolone-resistant, mutant strains of C. perfringens, with stable mutations either in DNA gyrase or in both DNA gyrase and topoisomerase IV, was compared with that produced by the wild-type parent strains to investigate the effect of mutations associated with the selection of gatifloxacin resistance on bacterial metabolic activities. The mutants differed from their respective wild-type parent strains in the enzymatic activities of azoreductase, nitroreductase, and beta-glucosidase and in the ratio of butyric acid to acetic acid production. Microarray analysis of one wild type and the corresponding mutant revealed different levels of mRNA expression for the enzymes involved in short-chain fatty acid (SCFA) synthesis and for beta-glucosidase and oxidoreductases. In addition to mutations in the target genes, selection of resistance to gatifloxacin resulted in strain-specific physiological changes in the resistant mutants of C. perfringens that affected their metabolic activities.

Novel Approach for Comparing the Abilities of Quinolones To Restrict the Emergence of Resistant Mutants during Quinolone Exposure

An agar-plate assay was adapted to examine aspects of quinolone structure that restrict the emergence of quinolone-mediated quinolone resistance. When Escherichia coli was applied to agar containing nalidixic acid, the number of quinolone-resistant mutants arising during incubation was decreased by raising the drug concentration and by mutations expected to block the induction of the SOS response (recA, lexA); the mutant number was increased by a mutator mutation (ung). The examination of four related fluoroquinolones then revealed that a C-8 methoxy group and an N-ethyl piperazine substituent at C-7 reduced mutant acquisition more effectively than C-8 H and C-7 C-ethyl piperazine groups. The fluoroquinolone that was most effective at restricting mutant acquisition was the most active when lethal activity was measured on agar plates or in liquid medium (as minimal bactericidal concentration). It also exhibited the lowest ratio of mutant MIC to wild-type MIC when it was tested with a set of isogenic gyrase mutants, and it had a low mutant prevention concentration (MPC) relative to MIC. However, a low MPC was less likely to be important in restricting the induced mutant accumulation because a fluoroquinolone N-ethyl piperazine substituent was more effective than a C-ethyl piperazine substituent at reducing mutant accumulation but was less effective at lowering the MPC. An 8-methoxy-quinazoline-2,4-dione was also effective at restricting the accumulation of resistant mutants on agar. Collectively, these data characterize a simple assay for detection of drug-mediated resistance that is sensitive to the structures of GyrA inhibitors. The assay provides a new method for screening quinolones and quinolone-like molecules that complements MPC-based tests for restricting the emergence of resistance.

Emergence of a new mutation and its accumulation in the topoisomerase IV gene confers high levels of resistance to fluoroquinolones in Escherichia coli isolates

Mutations in DNA gyrase and topoisomerase IV genes are the main mechanisms of resistance to quinolones. In this study, we determined mutations in gyrA, gyrB, parC and parE among 57 ciprofloxacin-resistant Escherichia coli isolates from a South Korean hospital and analysed the relationship between the minimal inhibitory concentrations (MICs) of fluoroquinolones and mutations in the topoisomerase IV gene. All ciprofloxacin-resistant E. coli isolates carried double mutations in gyrA and at least a single mutation in parC; some isolates also carried a single mutation in parE. The most common mutations were S83L and D87N in g yrA, S80I in parC and S458A in parE, which accounted for 25% of isolates. Single mutations in parE at L445I, S458P and S458W were identified for the first time. Double mutations in parC and a combination of single mutations in parC and parE significantly increased the MIC values of fluoroquinolones. In vitro induction of resistance to ciprofloxacin showed that double mutations in gyrA were a prerequisite to conferring a resistant phenotype to fluoroquinolones, and an additional mutation in the topoisomerase IV gene increased the MIC values of ciprofloxacin. In conclusion, emergence of a new mutation in parC and parE and its accumulation induces high levels of resistance to fluoroquinolones in E. coli.

Salmonella entericaSerovar Typhi with CTX-M β-Lactamase, Germany

<i>Salmonella enterica</i>Serovar Typhi with CTX-M β-Lactamase, Germany

Emerging fluoroquinolone-non-susceptible group A streptococci in two different paediatric populations

Clonal emergence of group A streptococci (GAS) with reduced susceptibility to fluoroquinolones (FQs) has been increasingly reported. Non-susceptibility is associated with various point mutations in the target-encoding genes and has only been described in a few emm types. We used a well-characterised GAS clinical paediatric collection from Brussels (Belgium) and Brasília (Brazil) to analyse the molecular basis of FQ non-susceptibility. GAS strains were tested for ciprofloxacin susceptibility and were screened for mutations in DNA gyrase- and topoisomerase IV-encoding genes. Genetic relationships between the different emm types were assessed by phylogenetic analysis of the whole surface-exposed part of the M protein. A high proportion (22.5%) of ciprofloxacin-non-susceptible isolates (minimal inhibitory concentration ≥2 mg/L) was found among the Belgian strains. They belonged mostly to emm type 6 (87%). In Brazil, 6% of the isolates, belonging to seven distantly related emm types, were non-susceptible. Our phylogenetic analysis showed that non-susceptibility may arise in various genetic backgrounds. Sequence comparison of the quinolone resistance-determining regions (QRDRs) of the ParC- and ParE-encoding genes from susceptible and non-susceptible isolates revealed that most of the mutations were found in both classes of isolates, indicating an emm type-linked polymorphism. In conclusion, we observed a clonal spreading of non-susceptible emm type 6 GAS strains in Brussels and a polyclonal distribution of non-susceptible isolates in Brazil. All the Brazilian and Belgian emm type 6 strains displayed a S79A/F mutation in parC that convincingly explains the non-susceptible phenotype.

Mutation in DNA Gyrase and Topoisomerase IV of V. cholerae Causing Diminished Susceptibility to Ciprofloxacin

Mutation in DNA Gyrase and Topoisomerase IV of V. cholerae Causing Diminished Susceptibility to Ciprofloxacin

Effect of transcriptional activators RamA and SoxS on expression of multidrug efflux pumps AcrAB and AcrEF in fluoroquinolone-resistant Salmonella Typhimurium

Multidrug resistance (MDR) including fluoroquinolone resistance in Salmonella Typhimurium can result from overexpression of efflux pumps. We examined the mechanisms of fluoroquinolone resistance among in vitro-induced ciprofloxacin-resistant Salmonella Typhimurium mutants, LTL and LTH, derived from laboratory strain LT2. Deletion mutation and RT-PCR techniques were employed to study the role of efflux pumps in fluoroquinolone resistance and their regulation cascades. In addition to point mutations in DNA gyrase (gyrA, gyrB) and topoisomerase IV (parC, parE) genes, increased expression of efflux pump genes, such as acrAB and acrEF, was observed in fluoroquinolone-resistant Salmonella strains. Constitutive expression of ramA containing a 9 bp deletion in the promoter region was directly associated with the overexpression of acrAB and acrEF and conferred an MDR phenotype in LTL. Inactivation of ramA increased the antimicrobial susceptibility of LTL, whereas complementation with the mutant allele induced an MDR phenotype in drug-susceptible Salmonella Typhimurium LT2, as demonstrated by 2- to 64-fold increases in resistance to fluoroquinolones, tetracycline and chloramphenicol. On the other hand, inactivation of mutant soxRS resulted in a slight increase in the susceptibility of LTH to several fluoroquinolone drugs, and the introduction of the mutant allele had no effect on antimicrobial susceptibility of LT2, indicating that constitutive expression of soxRS played a minimum role in fluoroquinolone resistance. Mutations in the promoter region of ramA appear to play a role in the up-regulation of RamA and AcrAB, and RamA is an activator of the MDR regulation cascade in Salmonella Typhimurium.

Clerocidin selectively modifies the gyrase-DNA gate to induce irreversible and reversible DNA damage

Clerocidin (CL), a microbial diterpenoid, reacts with DNA via its epoxide group and stimulates DNA cleavage by type II DNA topoisomerases. The molecular basis of CL action is poorly understood. We establish by genetic means that CL targets DNA gyrase in the Gram-positive bacterium Streptococcus pneumoniae, and promotes gyrase-dependent single- and double-stranded DNA cleavage in vitro. CL-stimulated DNA breakage exhibited a strong preference for guanine preceding the scission site (−1 position). Mutagenesis of −1 guanines to A, C or T abrogated CL cleavage at a strong pBR322 site. Surprisingly, for double-strand breaks, scission on one strand consistently involved a modified (piperidine-labile) guanine and was not reversed by heat, salt or EDTA, whereas complementary strand scission occurred at a piperidine-stable −1 nt and was reversed by EDTA. CL did not induce cleavage by a mutant gyrase (GyrA G79A) identified here in CL-resistant pneumococci. Indeed, mutations at G79 and at the neighbouring S81 residue in the GyrA breakage-reunion domain discriminated poisoning by CL from that of antibacterial quinolones. The results suggest a novel mechanism of enzyme inhibition in which the −1 nt at the gyrase-DNA gate exhibit different CL reactivities to produce both irreversible and reversible DNA damage.