Mutations In Topoisomerase Research Articles

Gyrase and Topoisomerase IV: Recycling Old Targets for New Antibacterials to Combat Fluoroquinolone Resistance.

Beyond their requisite functions in many critical DNA processes, the bacterial type II topoisomerases, gyrase and topoisomerase IV, are the targets of fluoroquinolone antibacterials. These drugs act by stabilizing gyrase/topoisomerase IV-generated DNA strand breaks and by robbing the cell of the catalytic activities of these essential enzymes. Since their clinical approval in the mid-1980s, fluoroquinolones have been used to treat a broad spectrum of infectious diseases and are listed among the five "highest priority" critically important antimicrobial classes by the World Health Organization. Unfortunately, the widespread use of fluoroquinolones has been accompanied by a rise in target-mediated resistance caused by specific mutations in gyrase and topoisomerase IV, which has curtailed the medical efficacy of this drug class. As a result, efforts are underway to identify novel antibacterials that target the bacterial type II topoisomerases. Several new classes of gyrase/topoisomerase IV-targeted antibacterials have emerged, including novel bacterial topoisomerase inhibitors, Mycobacterium tuberculosis gyrase inhibitors, triazaacenaphthylenes, spiropyrimidinetriones, and thiophenes. Phase III clinical trials that utilized two members of these classes, gepotidacin (triazaacenaphthylene) and zoliflodacin (spiropyrimidinetrione), have been completed with positive outcomes, underscoring the potential of these compounds to become the first new classes of antibacterials introduced into the clinic in decades. Because gyrase and topoisomerase IV are validated targets for established and emerging antibacterials, this review will describe the catalytic mechanism and cellular activities of the bacterial type II topoisomerases, their interactions with fluoroquinolones, the mechanism of target-mediated fluoroquinolone resistance, and the actions of novel antibacterials against wild-type and fluoroquinolone-resistant gyrase and topoisomerase IV.

Phenotypic and genetic insights into efflux pump mechanism in Mycoplasma anserisalpingitidis.

Mycoplasma anserisalpingitidis is one of the most important waterfowl-pathogenic mycoplasmas. Due to inadequate antibiotic treatment, many strains with high minimal inhibitory concentration (MIC) values for multiple drugs have been isolated lately. Decreased antibiotic susceptibility in several Mycoplasma species are known to be associated with mutations in topoisomerase and ribosomal genes, but other strategies such as active efflux pump mechanisms were also described. The scope of this study was the phenotypic and genetic characterization of the active efflux mechanism in M. anserisalpingitidis. We measured the MIC values in the presence and absence of different efflux pump inhibitors (EPIs), such as carbonyl cyanide m-chlorophenylhydrazine (CCCP), orthovanadate (OV), and reserpine (RSP). Moreover, bioinformatic tools were utilized to detect putative regulatory sequences of membrane transport proteins coding genes, while comparative genome analysis was performed to reveal potential markers of antibiotic resistance. Out of the three examined EPIs, CCCP decreased the MICs at least two-fold below the original MICs (in 23 cases out of 36 strains). In the presence of OV or RSP, MIC value differences could be seen only if modified dilution series (10% decrease steps were used instead of two-fold dilutions) were applied (in 24/36 cases with OV and 9/36 with RSP). During comparative genome analysis, non-synonymous single nucleotide polymorphisms (nsSNPs) were identified in genes encoding ABC membrane transport proteins, which were displayed in higher percentages in M. anserisalpingitidis strains with increased MICs. In terms of other genes, a nsSNP was identified in DNA gyrase subunit A (gyrA) gene which can be related to decreased susceptibility to enrofloxacin. The present study is the first to highlight the importance of efflux pump mechanisms in M. anserisalpingitidis. Considering the observed effects of the EPI CCCP against this bacterium, it can be assumed, that the use of EPIs would increase the efficiency of targeted antibiotic therapy in the future control of this pathogen. However, further research is required to obtain a more comprehensive understanding of efflux pump mechanism in this bacterium.

Characterization of Mutations in DNA Gyrase and Topoisomerase IV in Field Strains and In Vitro Selected Quinolone-Resistant Mycoplasma hyorhinis Mutants.

Mycoplasma hyorhinis is ubiquitous in swine, and it is a common pathogen of swine that causes polyserositis, arthritis, and maybe pneumonia. Fluoroquinolones are effective antimicrobials used for the treatment of mycoplasmal infection. However, a decrease in fluoroquinolones susceptibility in mycoplasma was observed. The molecular mechanisms have been studied in many mycoplasma species, while the mechanism in M. hyorhinis is still unknown. This study aimed to illustrate the in vitro development of fluoroquinolone resistance in M. hyorhinis and unveil the resistance mechanisms in both in vitro selected mutants and field strains. Seven ciprofloxacin-sensitive M. hyorhinis isolates were chosen to induce the fluoroquinolone resistance in vitro, and the point mutations in the quinolone resistance-determining regions (QRDRs) were characterized. The substitutions first occurred in ParC, resulting in a 2- to 8-fold increase in resistance, followed by additional mutations in GyrA and/or ParE to achieve a 32-fold increase. The mutations occurred in hot spots of QRDRs, and they were diverse and variable, including five in ParC (Ser80Phe, Ser80Tyr, Phe80Tyr, Glu84Gly, and Glu84Lys), four in GyrA (Ala83Val, Ser84Pro, Asp87Tyr, and Asp87Asn) and one in ParE (Glu470Lys). Target mutations in field strains were observed in the ParC (Ser80Phe, Ser81Pro, and Glu84Gln) of isolates with MICCIP = 2 μg/mL. This study characterized the point mutations in the QRDRs of M. hyorhinis and could be useful for the rapid detection of fluoroquinolone resistance in M. hyorhinis field isolates.

SUMO: A Swiss Army Knife for Eukaryotic Topoisomerases.

Topoisomerases play crucial roles in DNA metabolism that include replication, transcription, recombination, and chromatin structure by manipulating DNA structures arising in double-stranded DNA. These proteins play key enzymatic roles in a variety of cellular processes and are also likely to play structural roles. Topoisomerases allow topological transformations by introducing transient breaks in DNA by a transesterification reaction between a tyrosine residue of the enzyme and DNA. The cleavage reaction leads to a unique enzyme intermediate that allows cutting DNA while minimizing the potential for damage-induced genetic changes. Nonetheless, topoisomerase-mediated cleavage has the potential for inducing genome instability if the enzyme-mediated DNA resealing is impaired. Regulation of topoisomerase functions is accomplished by post-translational modifications including phosphorylation, polyADP-ribosylation, ubiquitylation, and SUMOylation. These modifications modulate enzyme activity and likely play key roles in determining sites of enzyme action and enzyme stability. Topoisomerase-mediated DNA cleavage and rejoining are affected by a variety of conditions including the action of small molecules, topoisomerase mutations, and DNA structural forms which permit the conversion of the short-lived cleavage intermediate to persistent topoisomerase DNA–protein crosslink (TOP-DPC). Recognition and processing of TOP-DPCs utilizes many of the same post-translational modifications that regulate enzyme activity. This review focuses on SUMOylation of topoisomerases, which has been demonstrated to be a key modification of both type I and type II topoisomerases. Special emphasis is placed on recent studies that indicate how SUMOylation regulates topoisomerase function in unperturbed cells and the unique roles that SUMOylation plays in repairing damage arising from topoisomerase malfunction.

WCK 1152, WCK 1153: Discovery and structure activity relationship for the treatment of resistant pneumococcal and staphylococcal respiratory infections

Novel antibacterial agents needed constantly to counter the ever emergent resistance development to commercially available drugs; one of the effective synthetic antibacterial classes is fluoroquinolone (FQ). This study includes structure activity relationship based design and synthesis of novel fluoroquinolone molecules active against resistant pathogens bearing mutations of DNA gyrase and/or topoisomerase IV which also express efflux pumps. Here, series of compounds were prepared by treating 1-cyclopropyl-6,7-difluoro-8-methoxy-1,4-dihydro-4-oxo-quinoline-3-carboxylic acid as a core with various 4-substituted-3,3-dialkyl piperidines as side chains, through conventional synthetic approaches. Subsequently, antibacterial activities of these fluoroquinolones were examined against Streptococcus pneumoniae, SPN 5844 (Moxi resistant DNA gyrase and topo IV mutant) and SPN 706 (FQ efflux positive). The current manuscript covers >50 examples of fluoroquinolone NCEs, amongst 20 NCEs have shown MIC in the range of (0.4 to >6.25 μg/ml) for SPN 5844 and (0.1–12.5 μg/ml) for SPN 706 strains. During the course of this study; WCK 919, comprising two chiral isomers; WCK 1152 and WCK 1153 were emerged as lead among the different series synthesized. Advance studies suggested either WCK 1152 or WCK 1153 are the worthy candidates for further clinical developments for respiratory infections caused by resistant pneumococci and staphylococci. However, on the basis of in house preclinical work, WCK 1152 had been selected for phase-1 domestic clinical trials.

Interplay between IncF plasmids and topoisomerase mutations conferring quinolone resistance in the Escherichia coli ST131 clone: stability and resistance evolution.

The Escherichia coli ST131 H30-Rx subclone vehicles CTX-M-15 plasmids and mutations in gyrA and parC conferring multidrug resistance successfully in the clinical setting. The aim of this study was (1) to investigate the relationship of specific topoisomerase mutations on the stability of IncF (CTX-M producing) plasmids using isogenic E. coli mutants and (2) to investigate the impact of the IncF-type plasmids present in the E. coli clone ST131 on the evolution of quinolone resistance. E. coli ATCC 25922 (background strain) and derived mutants encoding specific QRDR substitutions were used. Also, NGS-characterized IncFIA and IncFIB plasmids (encoding CTX-M genes) were included. Plasmid stability was evaluated by sequential dilutions into Luria broth medium without antibiotics for 7days. Mutant frequency to ciprofloxacin was also evaluated. Moderate differences in the IncF plasmids stability were observed among E. coli ATCC 25922 and isogenic mutants. Under our experimental conditions, the fluctuation of bacteria harboring plasmids was less than 0.5-log(10) in all cases. In the mutant frequency tests, it was observed that the presence of these IncF plasmids increased this value significantly (10-1000-fold). Quinolone resistance substitutions in gyrA or parC genes, frequently found associated with E. coli clone ST131, do not modify the stability of ST131-associated IncFIA and IncFIB plasmids under in vitro conditions. IncF-type plasmids present in E. coli clone ST131 facilitate the selection of resistance to quinolones. These results are consistent with the clinical scenario in which the combination of resistance to quinolones and beta-lactams is highly frequent in the E. coli clone ST131.

Validation of Pefloxacin for detection of fluoroquinolone (FQ) resistance among Salmonella Typhi with special reference to GyrB mutations.

Introduction. Fluoroquinolone (FQ) resistant Salmonella are classified as high priority pathogens by WHO. FQ resistance among Salmonella Typhi has emerged rapidly and is predominantly mediated by mutations in the topoisomerase genes gyrA, and parC. Mutations in GyrA result in classical FQ resistance (DCS-NAR) i.e. decreased susceptibility to ciprofloxacin (MIC of 0.12 to 0.5 µg ml-1) (DCS) and resistance to nalidixic acid (NAR). Previously a nalidixic acid disc test was proposed for detection of DCS. Recently isolates with non-classical FQ resistance caused by plasmid-mediated quinolone resistance (PMQR) and mutations in GyrB have emerged. These mechanisms also result in DCS but are nalidixic acid susceptible (NAS) and thus pose diagnostic challenges. CLSI and EUCAST have recommended use of 5 µg pefloxacin discs for detection of DCS in Salmonella.Hypothesis. The CLSI and EUCAST recommendations for use of 5 µg pefloxacin for detection of DCS has not been validated on typhoidal Salmonella and resistance mediated by GyrB mutation in Salmonella species.Aim. The aim of the present study was to validate the performance of the 5 µg pefloxacin discs to detect isolates of S. Typhi with DCS with special reference to GyrB mutations.Methodology. A total of 180 clinical isolates of Salmonella Typhi (2005-2014) were investigated for genetic mechanisms of resistance. Zone diameters for nalidixic acid (30μg), ciprofloxacin (5μg) and pefloxacin (5µg) and minimum inhibitory concentration (MIC) for ciprofloxacin were determined using CLSI guidelines. Performance of the three discs was evaluated to detect FQ resistance in S. Typhi.Results. Topoisomerase mutations in GyrB +/ ParC and GyrB were detected in 112 and 34 isolates respectively. Different mutations have a varied effect on the MIC for ciprofloxacin. The current breakpoints for susceptible (≤0.06 µg ml-1) and non-susceptible (≥0.125 µg ml-1), failed to detect all isolates with a resistance mechanism. Performance of both ciprofloxacin and pefloxacin discs were excellent compared to nalidixic acid in differentiating isolates with non-classical resistance mediated by GyrB from wild-type.Conclusion. The pefloxacin disc can be used to detect FQ resistance among S. Typhi. This is the first report of validation of pefloxacin for detection of FQ resistance in S. Typhi mediated by GyrB mutation.

Nybomycin inhibits both types of E. coli DNA gyrase - fluoroquinolone-sensitive and fluoroquinolone-resistant.

Bacterial type II topoisomerases, DNA gyrase and topoisomerase IV, are targets of many antibiotics including fluoroquinolones (FQs). Unfortunately, a number of bacterial species easily acquire resistance to FQs by mutations in either DNA gyrase or topoisomerase IV genes. The emergence of resistant pathogenic strains is a global problem in healthcare, therefore, identifying alternative pathways to thwart their persistence is the current frontier in drug discovery. An attractive class of compounds is nybomycins, reported to be "reverse antibiotics" that selectively inhibit growth of some Gram-positive FQ-resistant bacteria by targeting the mutant form of DNA gyrase, while being inactive against wild-type strains with FQ-sensitive gyrases. The strong "reverse" effect was demonstrated only for a few Gram-positive organisms resistant to FQs due to the S83L/I mutation in GyrA subunit of DNA gyrase. However, the activity of nybomycins has not been extensively explored among Gram-negative species. Here, we observed that in Gram-negative E. coli ΔtolC strain with enhanced permeability, wild-type gyrase and GyrA S83L mutant, resistant to fluoroquinolones, are both similarly sensitive to nybomycin.

Antibiotic prophylaxis and the gastrointestinal resistome in paediatric patients with acute lymphoblastic leukaemia: a cohort study with metagenomic sequencing analysis

SummaryBackgroundAlthough antibiotic prophylaxis with levofloxacin can reduce the risk of serious infection in immunocompromised patients, the potential contribution of prophylaxis to antibiotic resistance is a major drawback. We aimed to identify the effects of levofloxacin prophylaxis, given to paediatric patients with acute lymphoblastic leukaemia to prevent infections during induction chemotherapy, on antibiotic resistance in gastrointestinal microbiota after completion of induction and consolidation therapy.MethodsThis prospective, single-centre (St Jude Children’s Research Hospital, Memphis, TN, USA) cohort study included children (≤18 years) receiving therapy for newly diagnosed acute lymphoblastic leukaemia and who received either primary levofloxacin prophylaxis or no antibacterial prophylaxis (aside from Pneumocystis jirovecii prophylaxis with trimethoprim-sulfamethoxazole) and provided at least two stool samples, including one after completion of induction therapy. We used metagenomic sequencing to identify bacterial genes that confer resistance to fluoroquinolones, trimethoprim-sulfamethoxazole, or other antibiotics, and to identify point mutations in bacterial topoisomerases (gyrA, parC) that confer resistance to fluoroquinolones. We then used generalised linear mixed models to compare the prevalence and relative abundance of antibiotic resistance gene groups after completion of induction and consolidation therapy between participants who had received levofloxacin and those who received no prophylaxis.FindingsBetween Feb 1, 2012, and April 30, 2016, 118 stool samples (32 baseline, 49 after induction, and 37 after consolidation) were collected from 49 evaluable participants; of these participants, 31 (63%) received levofloxacin prophylaxis during induction therapy and 18 (37%) received no antibacterial prophylaxis. Over the course of induction therapy, there was an overall increase in the relative abundance of trimethoprim-sulfamethoxazole resistance genes (estimated mean fold change 5·9, 95% CI 3·6–9·6; p<0·0001), which was not modified by levofloxacin prophylaxis (p=0·46). By contrast, the prevalence of topoisomerase point mutations increased over the course of induction therapy in levofloxacin recipients (mean prevalence 10·4% [95% CI 3·2–25·4] after induction therapy vs 3·7% [0·2–22·5] at baseline) but not other participants (0% vs 0%; p<0·0001). There was no significant difference between prophylaxis groups with respect to changes in aminoglycoside, β-lactam, vancomycin, or multidrug resistance genes after completion of induction or consolidation therapy.InterpretationAnalysing the gastrointestinal resistome can provide insights into the effects of antibiotics on the risk of antibiotic-resistant infections. In this study, antibiotic prophylaxis with trimethoprim-sulfamethoxazole or levofloxacin during induction therapy for acute lymphoblastic leukaemia appeared to increase the short-term and medium-term risk of colonisation with bacteria resistant to these antibiotics, but not to other drugs. More research is needed to determine the longer-term effects of antibacterial prophylaxis on colonisation with antibiotic-resistant bacteria.FundingChildren’s Infection Defense Center at St Jude Children’s Research Hospital, American Lebanese Syrian Associated Charities, and National Institutes of Health.

Identification of two novel type II topoisomerase mutations in Enterococcus spp. isolated from a hospital in China

Type II topoisomerases, including DNA gyrase (GyrA) and topoisomerase IV (ParC), contribute to fluoroquinolone resistance in Enterococcus spp. This study investigated the mutational status of the quinolone resistance-determining regions (QRDRs) of GyrA and ParC in the clinical isolates of enterococci from a hospital in Baotou, China. We analyzed 110 enterococcal isolates, including 57 Enterococcus faecalis and 53 Enterococcus faecalis faecium. The resistance rates of E. faecalis and E. faecium to ciprofloxacin were 63.16% and 84.91%, respectively. We found that 32 samples of E. faecalis and 42 of E. faecium had single or combined mutations in gyrA and/or parC, which were all resistant to ciprofloxacin. Only two ciprofloxacin-resistant E. faecalis isolates had no mutation. No mutations in gyrA and parC genes in all ciprofloxacinsusceptible isolates were found. Ciprofloxacin minimal inhibitory concentrations (MICs) in the mutation group were significantly higher than those of the non-mutation group, indicating that mutations in the QRDRs of gyrA and parC were correlated with MIC elevation. Two novel substitutions (GyrA Ser83Phe and ParC Ser80Leu) of E. faecalis were identified herein. Three-dimensional modeling revealed that these novel amino acid substitutions could disrupt the water/metal-ion bridge and decrease the interaction between the enzymes and ciprofloxacin. The data showed a diversity of mutation types in QRDRs of type II topoisomerases whose association with fluoroquinolone resistance in clinical isolates of enterococci warrants further investigation.

Evaluation of Fluoroquinolone Resistance in Clinical Avian Pathogenic Escherichia coli Isolates from Flanders (Belgium).

Fluoroquinolones are frequently used antimicrobials for the treatment of avian pathogenic Escherichia coli (APEC) infections. However, rapid development and selection of resistance to this class of antimicrobial drugs is a significant problem. The aim of this study was to investigate the occurrence and mechanisms of antimicrobial resistance against enrofloxacin (ENRO) in APEC strains in Flanders, Belgium. One hundred and twenty-five APEC strains from broilers with clinical colibacillosis were collected in Flanders from November 2017 to June 2018. The minimum inhibitory concentration (MIC) of all strains and the mutant prevention concentration (MPC) of a sample of sensitive isolates were determined using a commercial gradient strip test and via the agar dilution method, respectively. Non-wild type (NWT) isolates were further characterized using polymerase chain reaction (PCR), gel electrophoresis and gene sequencing. Forty percent of the APEC strains were NWT according to the epidemiological cut-off (ECOFF) measure (MIC > 0.125 μg/mL). With respect to clinical breakpoints, 21% were clinically intermediate (0.5 ≤ MIC ≤ 1 μg/mL) and 10% were clinically resistant (MIC ≥ 2). The MPC values of the tested strains ranged from 0.064 to 1 μg/mL, resulting in MPC/MIC ratios varying from 4 to 32. The majority (92%) of the NWT strains carried one or two mutations in gyrA. Less than a quarter (22%) manifested amino acid substitutions in the topoisomerase IV parC subunit. Only three of the NWT strains carried a mutation in parE. Plasmid mediated quinolone resistance (PMQR) associated genes were detected in 18% of the NWT strains. In contrast to the relatively large number of NWT strains, only a small percentage of APEC isolates was considered clinically resistant. The most common MPC value for sensitive strains was 0.125 μg/mL. Some isolates showed higher values, producing wide mutant selection windows (MSW). Chromosomal mutations in DNA gyrase and topoisomerase IV were confirmed as the main source of decreased antimicrobial fluoroquinolone susceptibility, de-emphasizing the role of PMQR mechanisms.

Efflux pump activity potentiates the evolution of antibiotic resistance across S. aureus isolates

The rise of antibiotic resistance in many bacterial pathogens has been driven by the spread of a few successful strains, suggesting that some bacteria are genetically pre-disposed to evolving resistance. Here, we test this hypothesis by challenging a diverse set of 222 isolates of Staphylococcus aureus with the antibiotic ciprofloxacin in a large-scale evolution experiment. We find that a single efflux pump, norA, causes widespread variation in evolvability across isolates. Elevated norA expression potentiates evolution by increasing the fitness benefit provided by DNA topoisomerase mutations under ciprofloxacin treatment. Amplification of norA provides a further mechanism of rapid evolution in isolates from the CC398 lineage. Crucially, chemical inhibition of NorA effectively prevents the evolution of resistance in all isolates. Our study shows that pre-existing genetic diversity plays a key role in shaping resistance evolution, and it may be possible to predict which strains are likely to evolve resistance and to optimize inhibitor use to prevent this outcome.

Identification of Antimicrobial Resistance-Associated Genes through Whole Genome Sequencing of Mycoplasma bovis Isolates with Different Antimicrobial Resistances.

Antimicrobial resistance (AMR) in Mycoplasma bovis has been previously associated with topoisomerase and ribosomal gene mutations rather than specific resistance-conferring genes. Using whole genome sequencing (WGS) to identify potential new AMR mechanisms for M. bovis, it was found that a 2019 clinical isolate with high MIC (2019-043682) for fluoroquinolones, macrolides, lincosamides, pleuromutilins and tetracyclines had a new core genome multilocus sequencing (cgMLST) type (ST10-like) and 91% sequence similarity to the published genome of M. bovis PG45. Closely related to PG45, a 1982 isolate (1982-M6152) shared the same cgMLST type (ST17), 97.2% sequence similarity and low MIC results. Known and potential AMR- associated genetic events were identified through multiple sequence alignment of the three genomes. Isolate 2019-043682 had 507 genes with non-synonymous mutations (NSMs) and 67 genes disrupted. Isolate 1982-M6152 had 81 NSMs and 20 disruptions. Using functional roles and known mechanisms of antimicrobials, a 55 gene subset was assessed for AMR potential. Seventeen were previously identified from other bacteria as sites of AMR mutation, 38 shared similar functions to them, and 11 contained gene-disrupting mutations. This study indicated that M. bovis may obtain high AMR characteristics by mutating or disrupting other functional genes, in addition to topoisomerases and ribosomal genes.

Characterization of quinolone-resistant enterococcus faecalis isolates from healthy chickens and pigs in Taiwan

This study attempted to correlate quinolone-resistant phenotypes and genetic traits in Enterococcus faecalis from healthy chickens and pigs in Taiwan. The percentage of E. faecalis isolates resistant to ciprofloxacin was 54.0% (162/300) in pigs and 53.5% (107/200) in chickens. Two hundred and sixty-nine ciprofloxacin-resistant E. faecalis isolates showed different levels of resistance to ciprofloxacin (MIC 4-512 mg/L), enrofloxacin (MIC 8-512 mg/L) and moxifloxacin (MIC 0.5-512 mg/L). Two mutations associated with resistance were detected in GyrA at Ser83 (to Arg/Ile) and Glu87 (to Lys/Gly) and one mutation was found in ParC at position 80 (Ser to Ile). In addition, triple-point mutation in DNA gyrase (GyrA) and topoisomerase IV (ParC) of E. faecalis was firstly reported. Thirty-six isolates with no amino acid substitution in GyrA or ParC showed high levels of ciprofloxacin resistance. In the presence of reserpine, the levels of resistance to ciprofloxacin for these 36 strains were decreased. The effect of reserpine on ciprofloxacin resistance was correlated with the level of expression of the emeA gene. Our results demonstrate that not only point mutations in topoisomerase IV and DNA gyrase but also the efflux pump can be used by E. faecalis to generate resistance to quinolones.

DNA topoisomerases as targets of anticancer drugs

Eukaryotic DNA topoisomerases are ubiquitous nuclear enzymes that alter DNA topology by breaking and rejoining DNA strands. There are two classes of DNA topoisomerases; DNA topoisomerase I introduces a transient single-strand DNA break, while topoisomerase II introduces transient double-strand DNA breaks for each catalytic reaction. Both are important for solving topological problems arising during DNA replication, transcription, recombination and other cellular functions. Recently, scientists have realized the importance of topoisomerases as new therapeutic targets for antibacterial, antifungal, antiparasitic, antiviral and anticancer drugs. The present review focuses on anticancer drugs targeting mammalian DNA topoisomerases, which are named DNA topoisomerase poison. Camptothecin and VM26 (teniposide) are representative DNA topoisomerase poisons that target DNA topoisomerase I and topoisomerase II, respectively. These drugs alter the breakage-reunion reactions of DNA topoisomerases by trapping topoisomerase-DNA cleavable complexes in both the purified system and cultured cells. However, resistance to various DNA topoisomerase poisons has been documented in cancer cells with repect to MDR1 overexpression, reduced topoisomerase levels, drug-resistant mutant topoisomerase, lengthened cell cycle time and altered DNA repair function. A better understanding of the molecular targets for anticancer drugs and the various drug-resistance mechanisms may help us to discover and tailor new drugs for particular drug-resistant tumors.

Characterizing Antimicrobial Resistant Escherichia coli and Associated Risk Factors in a Cross-Sectional Study of Pig Farms in Great Britain.

Combatting antimicrobial resistant (AMR) using a One-Health approach is essential as various bacteria, including Escherichia coli, a common bacteria, are becoming increasingly resistant and livestock may be a reservoir. The AMR gene content of 492 E. coli, isolated from 56 pig farms across Great Britain in 2014–2015, and purified on antibiotic selective and non-selective plates, was determined using whole genome sequencing (WGS). The E. coli were phylogenetically diverse harboring a variety of AMR profiles with widespread resistance to “old” antibiotics; isolates harbored up to seven plasmid Inc-types. None showed concurrent resistance to third-generation cephalosporins, fluoroquinolones and clinically relevant aminoglycosides, although ∼3% harbored AMR genes to both the former two. Transferable resistance to carbapenem and colistin were absent, and six of 117 E. coli STs belonged to major types associated with human disease. Prevalence of genotypically MDR E. coli, gathered from non-selective media was 35% and that of extended-spectrum-beta-lactamase E. coli was low (∼2% from non-selective). Approximately 72.6% of E. coli from ciprofloxacin plates and only 8.5% from the other plates harbored fluoroquinolone resistance due to topoisomerase mutations; the majority were MDR. In fact, multivariable analysis confirmed E. coli purified from CIP enrichment plates were more likely to be MDR, and suggested MDR isolates were also more probable from farms with high antibiotic usage, specialist finisher farms, and farms emptying their manure pits only after each batch. Additionally, farms from the South East were more likely to have MDR E. coli, whereas farms in Yorkshire and the Humber were less likely. Future investigations will determine whether suggested improvements such as better biosecurity or lower antimicrobial use decreases MDR E. coli on pig farms. Although this study focuses on pig farms, we believe the methodology and findings can be applied more widely to help livestock farmers in the United Kingdom and elsewhere to tackle AMR.

Co-Incidence of Type II Topoisomerase Mutations and Efflux Expression in High Fluoroquinolone Resistant Enterococcus faecalis Isolated from Urinary Tract Infections.

IntroductionEnterococcus faecalis is one of the most common pathogens in urinary tract infections (UTIs). Fluoroquinolones have been frequently used to treat E. faecalis UTIs, and the emergence of fluoroquinolone-resistant E. faecalis strains has recently been reported in several countries. This study aimed to elucidate the mechanisms involved in fluoroquinolone resistance in clinical E. faecalis isolates by analyzing mutations in quinolone- resistance-determining regions (QRDRs) of gyrA and parC and investigating the role of some efflux pumps.MethodsIn total, 70 clinical E. faecalis isolates collected from UTIs were identified by phenotypic and genotypic methods. Antimicrobial susceptibility testing was performed and multidrug-resistant (including ciprofloxacin resistant) isolates were studied for minimum inhibitory concentrations to ciprofloxacin, levofloxacin, and ofloxacin. In the following, mutations in QRDRs of gyrA and parC and expression of EfrA, EfrB, and EmeA efflux pumps were investigated in 20 high-level ciprofloxacin resistant and two ciprofloxacin susceptible isolates.ResultsHigh-level resistance to ciprofloxacin was detected in 97.5% of isolates. Sequencing of QRDRs revealed that 65% and 75% of isolates carried mutations in gyrA and parC, respectively. The presence of efflux genes was detected in all studied isolates, but expression of efrA, emeA, and efrB was demonstrated in 50%, 40%, and 30% of resistant isolates, respectively. Neither QRDR mutation nor the expression of efflux genes showed any significant association with MIC.ConclusionCo-incidence of mutation and efflux gene expression in more than half of isolates (13/20) suggests that both mechanisms may play a role in fluoroquinolone resistance. The other unknown mechanisms including different efflux pumps and probably other QRDRs mutations may contribute to fluoroquinolone resistance in E. faecalis.

Point mutations in topoisomerase I alter the mutation spectrum in E. coli and impact the emergence of drug resistance genotypes.

Identifying the molecular mechanisms that give rise to genetic variation is essential for the understanding of evolutionary processes. Previously, we have used adaptive laboratory evolution to enable biomass synthesis from CO2 in Escherichia coli. Genetic analysis of adapted clones from two independently evolving populations revealed distinct enrichment for insertion and deletion mutational events. Here, we follow these observations to show that mutations in the gene encoding for DNA topoisomerase I (topA) give rise to mutator phenotypes with characteristic mutational spectra. Using genetic assays and mutation accumulation lines, we find that point mutations in topA increase the rate of sequence deletion and duplication events. Interestingly, we observe that a single residue substitution (R168C) results in a high rate of head-to-tail (tandem) short sequence duplications, which are independent of existing sequence repeats. Finally, we show that the unique mutation spectrum of topA mutants enhances the emergence of antibiotic resistance in comparison to mismatch-repair (mutS) mutators, and leads to new resistance genotypes. Our findings highlight a potential link between the catalytic activity of topoisomerases and the fundamental question regarding the emergence of de novo tandem repeats, which are known modulators of bacterial evolution.

Mutations in topoisomerase II\u03b2 result in a B cell immunodeficiency

B cell development is a highly regulated process involving multiple differentiation steps, yet many details regarding this pathway remain unknown. Sequencing of patients with B cell-restricted immunodeficiency reveals autosomal dominant mutations in TOP2B. TOP2B encodes a type II topoisomerase, an essential gene required to alleviate topological stress during DNA replication and gene transcription, with no previously known role in B cell development. We use Saccharomyces cerevisiae, and knockin and knockout murine models, to demonstrate that patient mutations in TOP2B have a dominant negative effect on enzyme function, resulting in defective proliferation, survival of B-2 cells, causing a block in B cell development, and impair humoral function in response to immunization.

A complex suite of loci and elements in eukaryotic type II topoisomerases determine selective sensitivity to distinct poisoning agents.

Type II topoisomerases catalyze essential DNA transactions and are proven drug targets. Drug discrimination by prokaryotic and eukaryotic topoisomerases is vital to therapeutic utility, but is poorly understood. We developed a next-generation sequencing (NGS) approach to identify drug-resistance mutations in eukaryotic topoisomerases. We show that alterations conferring resistance to poisons of human and yeast topoisomerase II derive from a rich mutational ‘landscape’ of amino acid substitutions broadly distributed throughout the entire enzyme. Both general and discriminatory drug-resistant behaviors are found to arise from different point mutations found at the same amino acid position and to occur far outside known drug-binding sites. Studies of selected resistant enzymes confirm the NGS data and further show that the anti-cancer quinolone vosaroxin acts solely as an intercalating poison, and that the antibacterial ciprofloxacin can poison yeast topoisomerase II. The innate drug-sensitivity of the DNA binding and cleavage region of human and yeast topoisomerases (particularly hTOP2β) is additionally revealed to be significantly regulated by the enzymes’ adenosine triphosphatase regions. Collectively, these studies highlight the utility of using NGS-based methods to rapidly map drug resistance landscapes and reveal that the nucleotide turnover elements of type II topoisomerases impact drug specificity.