Mutations In Topoisomerase Genes Research Articles

Evaluation of Resistance to Ciprofloxacin and Identification of Mutations in Topoisomerase Genes in Escherichia coli and Klebsiella pneumonia Isolated from Pediatric Urinary Tract Infections

Evaluation of Resistance to Ciprofloxacin and Identification of Mutations in Topoisomerase Genes in Escherichia coli and Klebsiella pneumonia Isolated from Pediatric Urinary Tract Infections

Drug resistance in topoisomerase-targeting therapy

Drug resistance is a well-known phenomenon that occurs when initially responsive to chemotherapy cancer cells become tolerant and elude further effectiveness of anticancer drugs. Based on their mechanism of action, anticancer drugs can be divided into cytotoxic-based agents and target-based agents. An important role among the therapeutics of the second group is played by drugs targeting topoisomerases, nuclear enzymes critical to DNA function and cell survival. These enzymes are cellular targets of several groups of anticancer agents which generate DNA damage in rapidly proliferating cancer cells. Drugs targeting topoisomerase I are mostly analogs of camtothecin, a natural compound isolated from the bark of a tree growing in China. Drugs targeting topoisomerase II are divided into poisons, such as anthracycline antibiotics, whose action is based on intercalation between DNA bases, and catalytic inhibitors that block topoisomerase II at different stages of the catalytic cycle. Unfortunately, chemotherapy is often limited by the induction of drug resistance. Identifying mechanisms that promote drug resistance is critical for the improvement of patient prognosis. Cancer drug resistance is a complex phenomenon that may be influenced by many factors. Here we discuss various mechanisms by which cancer cells can develop resistance to topoisomerase-directed drugs, which include enhanced drug efflux, mutations in topoisomerase genes, hypophosphorylation of topoisomerase II catalytic domain, activation of NF-κB transcription factor and drug inactivation. All these events may lead to the ineffective induction of cancer cell death. Attempts at circumventing drug resistance through the inhibition of cellular efflux pumps, use of silencing RNAs or inhibition of some important mechanisms, which can allow cancer cells to survive therapy, are also presented.

Chromosomal mutations that accompany qnr in clinical isolates of Escherichia coli

We examined 13 qnr-positive and 14 qnr-negative clinical isolates of Escherichia coli for mutations previously seen in a qnr-containing laboratory strain exposed to supra minimum inhibitory concentrations (MICs) of ciprofloxacin. Among the qnr-positive strains, those with ciprofloxacin MICs of ≥ 2 µg/mL had at least one mutation in gyrA. Mutations in parC were present in strains with a ciprofloxacin MIC of ≥ 128 µg/mL. The 6 most ciprofloxacin-resistant strains contained additional plasmid-mediated quinolone resistance determinants. aac(6')-Ib-cr was found in 5 of the 6 strains. Eleven of the 13 strains had alterations in MarR, 9 in SoxR, and 5 had mutations in AcrR. All had elevated expression of at least one efflux pump gene, predominantly acrA (92% of the strains), followed by mdtE (54%) and ydhE (46%). Nine had functionally silent alterations in rfa, two had mutations in gmhB, and one of these also had a mutation in surA. An E. coli with ciprofloxacin MIC of 1024 µg/mL contained 4 different plasmid-mediated quinolone resistance determinants as well as gyrA, parC, parE and pump overexpression mutations. Nine of the 14 qnr-negative strains had mutations in topoisomerase genes with a ciprofloxacin MIC of 0.25 to 256 µg/mL. The three most resistant strains also had mutations in parE. Twelve had alterations in MarR, 10 in SoxR and 5 in AcrR. Ten of the 14 strains had elevated expression of efflux pumps with acrA (71.4%), followed by ydhE (50%) and mdtE (14.3%). A diversity of resistance mechanisms occurs in clinical isolates with and without qnr genes.

The Impact of Mutations in Topoisomerase Genes and the Plasmid-Mediated Quinolone Resistance (PMQR) Determinants on the Resistance to Fluoroquinolones in Klebsiella pneumoniae

Background: Klebsiella pneumoniae causes a variety of infections including community acquired pneumonia and nosocomial infections. Objectives: The aim of this study was to determine the plasmid-associated quinolone-resistance (PMQR) genes and mutations in quinolone-resistance determining region (QRDR) of topoisomerase genes in K. pneumoniae. Methods: One hundred clinical K. pneumoniae isolates were collected from Kermanshah hospitals for this study. The minimum inhibitory concentrations (MIC) for levofloxacin and ciprofloxacin was determined by broth microdilution method. The presence of PMQR genes (qnrA, qnrB, qnrS, aac (6´)-Ib, and qepA) among isolates resistant to fluoroquinolones was detected by PCR. All PCR products for topoisomerase genes (gyrA, gyrB and parC) were sequenced and analyzed. Results: Sequence analysis showed mutations in gyrA at codons 83 and 87 in 24 (85.7%) and 20 (71.4%) of isolates, respectively. For parC mutations were detected at codons 80 and 84 in 15 (53.6%) and 7 (25%) isolates, respectively. At the outside of QRDR in gyrA and gyrB, mutations were also observed. The mutation was not detected in the QRDR of gyrB. The qnrB, qnrS, and aac (6')-Ib-cr were found in 1 (3.6%), 11 (39.3%) and 25 (89.3%) of isolates, respectively. Howevr, the qnrA and qepA genes were not detected among isolates. Conclusions: The resistance to fluoroquinolones is relatively high among K. pneumonia and mutations in QRDR of topoisomerase genes play an important role for this resistance. The plasmid-mediated genes also increase the level of resistance. The mutations outside the QRDR of topoisomerase genes also occur, but their exact role in resistance needs to be clarified.

Multiple transmissible genes encoding fluoroquinolone and third-generation cephalosporin resistance co-located in non-typhoidal Salmonella isolated from food-producing animals in China

The aim of this study was to identify genes conferring resistance to fluoroquinolones and extended-spectrum β-lactams in non-typhoidal Salmonella (NTS) from food-producing animals in China. In total, 31 non-duplicate NTS were obtained from food-producing animals that were sick. Isolates were identified and serotyped and the genetic relatedness of the isolates was determined by pulsed-field gel electrophoresis of XbaI-digested chromosomal DNA. Antimicrobial susceptibility was determined using Clinical and Laboratory Standards Institute methodology. The presence of extended-spectrum β-lactamase (ESBL) and fluoroquinolone resistance genes was established by PCR and sequencing. Genes encoded on transmissible elements were identified by conjugation and transformation. Plasmids were typed by PCR-based replicon typing. The occurrence and diversity of numerous different transmissible genes conferring fluoroquinolone resistance [qnrA, qnrD, oqxA and aac(6′)-Ib-cr] and ESBLs (CTX-M-27 and CTX-M-14), and which co-resided in different isolates and serovars of Salmonella, were much higher than in European countries. Furthermore, different plasmids encoded fluoroquinolone resistance (ca. 6kb) and β-lactam resistance (ca. 63kb) and these co-resided in isolates with mutations in topoisomerase genes (gyrA and parC) giving very resistant Salmonella. The presence of multidrug-resistant bacteria in food-producing animals in countries that export foodstuffs suggests that global transfer of antibiotic resistances from country to country on food is possible.

Chromosomal and plasmid-mediated fluoroquinolone resistance mechanisms among broad-spectrum-cephalosporin-resistant Escherichia coli isolates recovered from companion animals in the USA

To determine the prevalence of plasmid-mediated quinolone resistance (PMQR) determinants and investigate mutations in gyrase and topoisomerase genes that may contribute to increased fluoroquinolone resistance in canine and feline Escherichia coli isolates in the USA that displayed reduced susceptibility to extended-spectrum cephalosporins. This study was undertaken because previous epidemiological studies identified a potential correlation between extended-spectrum cephalosporins and fluoroquinolone resistance. Isolates (n = 54) with reduced susceptibility to ceftazidime or cefotaxime were screened by PCR for the presence of PMQR determinants and gyrase and topoisomerase genes were sequenced. Isolates were further characterized by conjugation and phylogenetic analyses. PMQR determinants aac(6')-Ib-cr, qnrS and qepA were identified in 30, 23 and 5 isolates, respectively. Multiple mutations were identified in the quinolone resistance-determining region, including the novel substitutions of Glu-84 → Ala and Leu-88 → Gln in ParC and Arg-432 → Ser and Glu-460 → Val in ParE. The isolate that exhibited the highest level of enrofloxacin resistance (MIC > 256 mg/L) had a double mutation in gyrA (Ser-83 → Leu and Asp-87 → Asn) and a triple mutation in parC (Ser-80 → Ile, Glu-84 → Gly and a novel mutation, Leu-88 → Gln). The presence of PMQR genes increased the ciprofloxacin MIC values 4-fold to 8-fold in transconjugants relative to the recipient strain. Approximately 39% of the isolates belonged to phylogenetic group D and 30% to group B2, which typically contain an increased number of virulence determinants compared with other groups. Novel mutations in topoisomerase genes and PMQR determinants aac(6')-Ib-cr, qnrS and qepA genes were detected among extended-spectrum β-lactamase-producing E. coli in the USA.

Mutations in the quinolone resistance-determining regions of gyrA and parC in Enterobacteriaceae isolates from Brazil.

Mutations in the quinolone resistance-determining regions (QRDR) in chromosomal gyrA and parC genes and fluoroquinolone susceptibility profiles were investigated in quinolone-resistant Enterobacteriaceae isolated from community and hospitalized patients in the Brazilian Southeast region. A total of 112 nalidixic acid-resistant enterobacterial isolates collected from 2000 to 2005 were investigated for mutations in the topoisomerases genes gyrA and parC by amplifying and sequencing the QRDR regions. Susceptibility to fluoroquinolones was tested by the agar dilution method. Amongst the 112 enterobacterial isolates, 81 (72.3%) were resistant to ciprofloxacin and 5 (4.5%) showed reduced susceptibility. Twenty-six (23.2%) were susceptible to ciprofloxacin. Several alterations were detected in gyrA and parC genes. Escherichia coli isolates (47.7%) showed double mutations in the gyrA gene and a single one in the parC gene. Two unusual aminoacid substitutions are reported, an Asp87-Asn in a Citrobacter freundii isolate with reduced susceptibility to fluoroquinolones and a Glu84-Ala in one E. coli isolate. Only a parC gene mutation was found in fluoroquinolone-susceptible Enterobacter aerogenes. None of the isolates susceptible to ciprofloxacin presented mutations in topoisomerase genes. This comprehensive analysis of QRDRs in gyrA and parC genes, covering commonly isolated Enterobacteriaceae in Brazil is the largest reported up to now.

Prevalence and characteristics of quinolone resistance in Escherichia coli in veal calves

Quinolone resistance is studied and reported increasingly in isolates from humans, food-producing animals and companion animals. Resistance can be caused by chromosomal mutations in topoisomerase genes, plasmid-mediated resistance genes, and active transport through efflux pumps. Cross sectional data on quinolone resistance mechanisms in non-pathogenic bacteria from healthy veal calves is limited. The purpose of this study was to determine the prevalence and characteristics of quinolone resistance mechanisms in Escherichia coli isolates from veal calves, after more than 20 years of quinolone usage in veal calves. MIC values were determined for all isolates collected as part of a national surveillance program on antimicrobial resistance in commensal bacteria in food-producing animals in The Netherlands. From the strains collected from veal calves in 2007 (n=175) all isolates with ciprofloxacin MIC≥0.125mg/L (n=25) were selected for this study, and screened for the presence of known quinolone resistance determinants. In this selection only chromosomal mutations in the topoisomerase type II and IV genes were detected. The number of mutations found per isolate correlated with an increasing ciprofloxacin MIC. No plasmid-mediated quinolone resistance genes were found. The contribution of efflux pumps varied from no contribution to a 16-fold increase in susceptibility. No correlation was found with the presence of resistance genes of other antimicrobial classes, even though all quinolone non-wild type isolates were resistant to 3 or more classes of antibiotics other than quinolones. Over twenty years of quinolone usage in veal calves in The Netherlands did not result in a widespread occurrence of plasmid-mediated quinolone resistance, limiting the transmission of quinolone resistance to clonal distribution.

Determination of the Efflux Pump-Mediated Resistance Prevalence in Pseudomonas aeruginosa, Using an Efflux Pump Inhibitor

In gram negative bacteria, fluoroquinolone resistance is acquired by target mutations in topoisomerase genes or by reducing the permeation of drugs due to the increase in expression of endogenous multidrug efflux pumps that expel structurally unrelated antimicrobial agents. An ongoing challenge is searching for new inhibitory substances in order to block efflux pumps and restore the antibiotic drugs susceptibility. In this research, we sought to investigate the interplay between ciprofloxacin and an efflux pump inhibitor (EPI), phenyl alanine arginyl beta-naphtylamide (PAbetaN), to determine the prevalence of efflux pump overexpression in clinical isolates of Pseudomonas aeruginosa. Ciprofloxacin was tested at different concentrations (256-0.25 microg/ml) with a fixed concentration of PAbetaN (50 microg/ml). The isolates susceptibility profiles were analyzed by disc diffusion and agar dilution methods using 10 antibiotic discs and 4 powders. It was found that in the presence of PAbetaN, resistance to ciprofloxacin was inhibited obviously and MIC values were decreased. The comparison between subgroups of P. aeruginosa isolates with different resistance profiles indicates that efflux pump overexpression (EPO) is present in 35% of ciprofloxacin resistant isolates with no cross resistance and in variable frequencies among isolates showing cross resistance to other tested antibiotics: gentamicin (31%), ceftazidime (29%), and imipenem (18%). Altogether, these results imply that PAbetaN maybe effective to restore the fluoroquinolone drugs susceptibility in clinical treatment procedures. Results also show that increased use of a fluoroquinolone drug such as ciprofloxacin can affect the susceptibility of P. aeruginosa to other different antipseudomonal agents.

Evaluation of Melting Curve Analysis for Screening the Most Prevalent Mutations in Topoisomerase Genes from Streptococcus pneumoniae

In a recently published article, Fukushima et al. ([3][1]) described a novel PCR-melting curve analysis (PCR-MCA) method for rapid screening of the most prevalent point mutations related to fluoroquinolone resistance in Streptococcus pneumoniae . In their study, they included 22 levofloxacin (LVX)-

Contribution of Target Gene Mutations and Efflux to Decreased Susceptibility of Salmonella enterica Serovar Typhimurium to Fluoroquinolones and Other Antimicrobials

The mechanisms involved in fluoroquinolone resistance in Salmonella enterica include target alterations and overexpression of efflux pumps. The present study evaluated the role of known and putative multidrug resistance efflux pumps and mutations in topoisomerase genes among laboratory-selected and naturally occurring fluoroquinolone-resistant Salmonella enterica serovar Typhimurium strains. Strains with ciprofloxacin MICs of 0.25, 4, 32, and 256 microg/ml were derived in vitro using serovar Typhimurium S21. These mutants also showed decreased susceptibility or resistance to many nonfluoroquinolone antimicrobials, including tetracycline, chloramphenicol, and several beta-lactams. The expression of efflux pump genes acrA, acrB, acrE, acrF, emrB, emrD, and mdlB were substantially increased (>or=2-fold) among the fluoroquinolone-resistant mutants. Increased expression was also observed, but to a lesser extent, with three other putative efflux pumps: mdtB (yegN), mdtC (yegO), and emrA among mutants with ciprofloxacin MICs of >or=32 microg/ml. Deletion of acrAB or tolC in S21 and its fluoroquinolone-resistant mutants resulted in increased susceptibility to fluoroquinolones and other tested antimicrobials. In naturally occurring fluoroquinolone-resistant serovar Typhimurium strains, deletion of acrAB or tolC increased fluoroquinolone susceptibility 4-fold, whereas replacement of gyrA double mutations (S83F D87N) with wild-type gyrA increased susceptibility>500-fold. These results indicate that a combination of topoisomerase gene mutations, as well as enhanced antimicrobial efflux, plays a critical role in the development of fluoroquinolone resistance in both laboratory-derived and naturally occurring quinolone-resistant serovar Typhimurium strains.

Fluoroquinolone treatment of experimental Salmonella enterica serovar Typhimurium DT104 infections in chickens selects for both gyrA mutations and changes in efflux pump gene expression

To determine the efficacy of enrofloxacin (Baytril) in chickens in eradicating three different resistance phenotypes of Salmonella enterica and to examine the resistance mechanisms of resulting mutants. In two separate replicate experiments (I and II), three strains of Salmonella enterica serovar Typhimurium DT104 [strain A, fully antibiotic-sensitive strain; strain B, isogenic multiple antibiotic-resistant (MAR) derivative of A; strain C, veterinary penta-resistant phenotype strain containing GyrA Phe-83], were inoculated into day-old chicks at approximately 10(3) cfu/bird. At day 10, groups of chicks (n =10) were given either enrofloxacin at 50 ppm in their drinking water for 5 days or water alone (control). Caecal contents were monitored for presence of Salmonella and colonies were replica plated to media containing antibiotics or overlaid with cyclohexane to determine the proportion of isolates with reduced susceptibility. The MICs of antibiotics and cyclohexane tolerance were determined for selected isolates from the chicks. Mutations in topoisomerase genes were examined by DHPLC and expression of marA, soxS, acrB, acrD and acrF by RT-PCR. In experiment I, but not II, enrofloxacin significantly reduced the numbers of strain A compared with the untreated control group. In experiment II, but not I, enrofloxacin significantly reduced the numbers of strain B. Shedding of strain C was unaffected by enrofloxacin treatment. Birds infected with strains A and B gave rise to isolates with decreased fluoroquinolone susceptibility. Isolates derived from strain A or B requiring >128 mg/L nalidixic acid for inhibition contained GyrA Asn-82 or Phe-83. Isolates inhibited by 16 mg/L nalidixic acid were also less susceptible to antibiotics of other chemical classes and became cyclohexane-tolerant (e.g. MAR). These studies demonstrate that recommended enrofloxacin treatment of chicks rapidly selects for strains with reduced fluoroquinolone susceptibility from fully sensitive and MAR strains. It can also select for MAR isolates.

Clonal spread of fluoroquinolone non-susceptible Streptococcus pyogenes

Fluoroquinolones are an important group of antibiotics widely used in adults, and, despite the absence of official approval, these drugs are also used in children. So far, resistance to fluoroquinolones in Streptococcus pyogenes is very rare. During a national surveillance programme in Belgium from 1999 to 2002, 2793 non-duplicate S. pyogenes recovered from tonsillopharyngitis patients were screened for fluoroquinolone resistance. Mutations in topoisomerase genes and the presence of any efflux pump activity were investigated to elucidate the fluoroquinolone resistance mechanisms. Clonality was assessed by pulsed-field gel electrophoresis (PFGE) and emm typing. Non-susceptibility to fluoroquinolones, defined as ciprofloxacin MIC > or = 2 mg/L, was identified in 152 (5.4%) of 2793 S. pyogenes. Fifty-five (36%) fluoroquinolone non-susceptible isolates were investigated for known resistance mechanisms; all showed mutations in parC, and 29 (19%) isolates also in parE; antibiotic efflux was not noted. Two major PFGE types comprised 88% of fluoroquinolone non-susceptible S. pyogenes and belonged to serotypes emm6 and emm75. Overall, emm6 and emm75 constituted >90% of all fluoroquinolone non-susceptible isolates and showed a significant temporal and geographical shift within Belgian provinces. Although fluoroquinolone-susceptible S. pyogenes also showed fluctuations in the predominant S. pyogenes serotypes, emm6 or emm75 were under-represented in this population. Approx. 55% of the fluoroquinolone non-susceptible isolates were recovered from children ( < or =16 years). We show here, for the first time, a multi-clonal spread of fluoroquinolone non-susceptible S. pyogenes exhibiting a known resistance mechanism. Non-susceptibility to fluoroquinolones in paediatric isolates is of concern.

Mutations in Topoisomerase Genes of Fluoroquinolone-Resistant Salmonellae in Hong Kong

A total of 88 salmonella isolates (72 clinical isolates for which the ciprofloxacin MIC was >0.06 microg/ml, 15 isolates for which the ciprofloxacin MIC was < or =0.06 microg/ml, and Salmonella enterica serotype Typhimurium ATCC 13311) were studied for the presence of genetic alterations in four quinolone resistance genes, gyrA, gyrB, parC, and parE, by multiplex PCR amplimer conformation analysis. The genetic alterations were confirmed by direct nucleotide sequencing. A considerable number of strains had a mutation in parC, the first to be reported in salmonellae. Seven of the isolates sensitive to 0.06 micro g of ciprofloxacin per ml had a novel mutation at codon 57 of parC (Tyr57-->Ser) which was also found in 29 isolates for which ciprofloxacin MICs were >0.06 micro g/ml. Thirty-two isolates had a single gyrA mutation (Ser83-->Phe, Ser83-->Tyr, Asp87-->Asn, Asp87-->Tyr, or Asp87-->Gly), 34 had both a gyrA mutation and a parC mutation (29 isolates with a parC mutation of Tyr57-->Ser and 5 isolates with a parC mutation of Ser80-->Arg). Six isolates which were isolated recently (from 1998 to 2001) were resistant to 4 micro g of ciprofloxacin per ml. Two of these isolates had double gyrA mutations (Ser83-->Phe and Asp87-->Asn) and a parC mutation (Ser80-->Arg) (MICs, 8 to 32 microg/ml), and four of these isolates had double gyrA mutations (Ser83-->Phe and Asp87-->Gly), one parC mutation (Ser80-->Arg), and one parE mutation (Ser458-->Pro) (MICs, 16 to 64 micro g/ml). All six of these isolates and those with a Ser80-->Arg parC mutation were S. enterica serotype Typhimurium. One S. enterica serotype Typhi isolate harbored a single gyrA mutation (Ser83-->Phe), and an S. enterica serotype Paratyphi A isolate harbored a gyrA mutation (Ser83-->Tyr) and a parC mutation (Tyr57-->Ser); both of these isolates had decreased susceptibilities to the fluoroquinolones. The MICs of ciprofloxacin, levofloxacin, and sparfloxacin were in general the lowest of those of the six fluoroquinolones tested. Isolates with a single gyrA mutation were less resistant to fluoroquinolones than those with an additional parC mutation (Tyr57-->Ser or Ser80-->Arg), while those with double gyrA mutations were more resistant.

Fluoroquinolone resistance in Campylobacter species from man and animals: detection of mutations in topoisomerase genes.

Consecutive isolates of quinolone-resistant campylobacter isolated over a 5 year period (1990-1995) from the faeces of patients with enteritis in Plymouth, UK, were examined for the epidemiology of mutations in gyrA (n = 127). In addition, clinical isolates and poultry isolates from Germany, The Netherlands and other regions of the UK collected before 1995 were examined for mutations in the quinolone resistance-determining region of gyrA by single-stranded conformational polymorphism analysis and direct sequencing of a 270 bp fragment of PCR-generated DNA. The majority of isolates (173/208) carried a mutation at codon 86 in gyrA resulting in substitution of Ile for Thr; all of these were resistant to ciprofloxacin (MIC > or = 2 mg/L). One isolate of Campylobacter jejuni had a mutation at Asp-90, and another had a double mutation at Thr-86 and Pro-104. Only two resistant isolates showed no mutation in gyrA. A novel gyrA sequence was amplified from two Campylobacter lari and one C. jejuni, which exhibited a valine at codon 86. Only 8/192 isolates had changes in gyrB; all were shown to relate to silent mutations in gyrB and presumably reflect natural polymorphisms in the gene.