gyrB Mutations Research Articles

Association between mutations in GYR A/B and PAR C/E genes of Acinetobacter Baumannii clinical isolates and Fluoroquinolones resistance

Background: Acinetobacter baumannii (A. baumannii) is an important organism in hospital acquired infections. A. baumannii has emerged as Quinolones resistant organism.The maximum crucial mechanism of fluoroquinolones resistance is the mutation in genes that encode the subunits of DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE). Objectives: determination of mutations of gyrA/B and parC/E genes using Restriction Fragment Length Polymorphism PCR (RFLP - PCR) among fluoroquinolones resistant clinical isolates of A. baumannii showed in ICU patients at Al-Azhar University hospitals. Subjects & Methods: The study was conducted on 100 isolates of A.baumannii which subjected to molecular study of mutations of gyrA, gyrB, parC and parE genes beside determination of minimal inhibitory concentration (MIC). Results: The most A. baumannii isolates had MIC >128 µg/ml (61.1%). The resistant isolates showed combind mutations in both gyrA& parC genes most frequently (79.2%) then gyrA (9.7%), parC (8.3%) and gyrB (2.8%). Conclusion: Mutations in gyrA and parC are the most common in fluoroquinolones resistant A. baumannii isolated from Al-Azhar University hospitals

New dual ATP-competitive inhibitors of bacterial DNA gyrase and topoisomerase IV active against ESKAPE pathogens.

The rise in multidrug-resistant bacteria defines the need for identification of new antibacterial agents that are less prone to resistance acquisition. Compounds that simultaneously inhibit multiple bacterial targets are more likely to suppress the evolution of target-based resistance than monotargeting compounds. The structurally similar ATP binding sites of DNA gyrase and topoisomerase Ⅳ offer an opportunity to accomplish this goal. Here we present the design and structure-activity relationship analysis of balanced, low nanomolar inhibitors of bacterial DNA gyrase and topoisomerase IV that show potent antibacterial activities against the ESKAPE pathogens. For inhibitor 31c, a crystal structure in complex with Staphylococcus aureus DNA gyrase B was obtained that confirms the mode of action of these compounds. The best inhibitor, 31h, does not show any invitro cytotoxicity and has excellent potency against Gram-positive (MICs: range, 0.0078-0.0625μg/mL) and Gram-negative pathogens (MICs: range, 1-2μg/mL). Furthermore, 31h inhibits GyrB mutants that can develop resistance to other drugs. Based on these data, we expect that structural derivatives of 31h will represent a step toward clinically efficacious multitargeting antimicrobials that are not impacted by existing antimicrobial resistance.

Performance comparison of GenoTypeMTBDRs/Version 1 and version 2 for rapid detection of resistance to fluoroquinolones and injectable drugs among multidrug-resistant mycobacterium tuberculosis in a low MDR-TB setting

Aims and objectives: Fluoroquinolones (FQs) are potent antibiotics against multidrug-resistant Mycobacterium tuberculosis (MDR-TB) and resistance to FQs is associated with higher patient mortality. Rapid and accurate drug susceptibility testing (DST) against FQs and injectable drugs is essential for successful treatment of MDR-TB. FQ resistance mutation detection is equivalent to culture-based DST for predicting MDR-TB treatment outcome. This study compared performance of GenoTypeMTBDRsl Version 1 and Version 2 for detection of resistance to FQs and injectable drugs among MDR-TB strains in Kuwait, a country with low incidence of MDR-TB. Methods: MDR-TB strains (n=93) cultured from 74 pulmonary and 19 extra-pulmonary specimens from 93 patients were analyzed by GenoTypeMTBDRsl Version 1 and Version 2 for FQ and injectable drug resistance-associated mutation detection. Repeat isolates from 22 patients, 50 pansusceptible isolates and M. tuberculosis H37 Rv (as control) were also tested. Results were confirmed by PCR-sequencing of gyrA, gyrB, rrs, embB and eis genes. Fingerprinting of isolates was done by spoligotyping. Results: Among 93 MDR-TB strains, GenoTypeMTBDRsl Version 1 with/without PCR-sequencing identified 10 isolates with gyrA mutation (A90V, n=2; D94G, n=4; D94A, n=2; D94N, n=1; D94Y, n=1), seven isolates with rrs mutation (A1401G) and 51 isolates with an embB mutation. GenoTypeMTBDRsl Version 2 with/without PCR-sequencing confirmed Version 1 results for gyrA/rrs genes and additionally identified two isolates with gyrB mutation (T500N, n=1; N538D, n=1) and one isolate with eis mutation (-10 G/A). Repeat isolates yielded expected results. No mutation was detected in 50 pansusceptible isolates. Spoligotyping identified 10 Shared International Types among 12 isolates with gyrA/B mutation. Conclusions: Our data show that GenoTypeMTBDRsl Version 2 is superior to Version 1 as it identified two additional isolates with gyrB mutations. Improved detection of FQ resistance by GenoTypeMTBDRsl Version 2 will help in proper management of MDR-TB patients in low MDR-TB settings. Supported by KURS grant MI01/18.

Molecular characterization of mutations associated with resistance to second line drugs in Mycobacterium tuberculosis patients from Casablanca, Morocco.

ABSTRACTThe emergence and spread of extensively drug-resistant tuberculosis (XDR-TB) is a serious threat to global health. Therefore, its rapid diagnosis is crucial. The present study aimed to characterize mutations conferring resistance to second line drugs (SLDs) within multidrug Mycobacterium tuberculosis (MDR-MTB) isolates and to estimate the occurrence of XDR-TB in Casablanca, Morocco. A panel of 200 MDR-TB isolates was collected at the Pasteur Institute between 2015-2018. Samples were subjected to drug susceptibility testing to Ofloxacin (OFX), Kanamycin (KAN) and Amikacin (AMK). The mutational status of gyrA, gyrB, rrs, tlyA and eis was assessed by sequencing these target genes. Drug susceptibility testing for SLDs showed that among the 200 MDR strains, 20% were resistant to OFX, 2.5% to KAN and 1.5% to AMK. Overall, 14.5% of MDR strains harbored mutations in gyrA, gyrB, rrs and tlyA genes. From the 40 OFXR isolates, 67.5% had mutations in QRDR of gyrA and gyrB genes, the most frequent one being Ala90Val in gyrA gene. Of note, none of the isolates harbored simultaneously mutations in gyrA and gyrB genes. In eight out of the 200 MDR-TB isolates resistant either to KAN or AMK, only 25% had A1401G or Lys89Glu change in rrs and tlyA genes respectively. This study is very informative and provides data on the alarming rate of fluoroquinolone resistance which warrants the need to implement appropriate drug regimens to prevent the emergence and spread of more severe forms of Mycobacterium tuberculosis drug resistance.

Fluoroquinolone resistance of Staphylococcus epidermidis isolated from healthy conjunctiva and analysis of their mutations in quinolone-resistance determining region

BackgroundStaphylococcus epidermidis is the most common pathogen in postoperative endophthalmitis and causes various infectious eye diseases. However, there is very little information on fluoroquinolone antibiotic resistance to S. epidermidis identified in conjunctival microbe and analysis of related genes. Here, the authors investigated the rate of resistance to fluoroquinolones of Staphylococcus epidermidis isolated from normal conjunctival microbes and mutations in the quinolone-resistance determining region (QRDR).Methods377 eye samples from 187 patients who underwent intravitreal injection and cataract surgery were included. Specimens were taken from the bilateral lower conjunctival sacs using a cotton swab and cultured. The cultures were identified using MALDI-TOP MS and gyrA, gyrB, parC, and parE gene mutations of QRDR were confirmed by DNA extraction from resistant strains of S. epidermidis with a micro-dilution method using ciprofloxacin, levofloxacin, and moxifloxacin.ResultsThe culture positive rate was 61.8% (231) for 374 eye samples. Of the 303 total strains cultured, S. epidermidis was the most common with 33.7% (102). Ten types of gene mutations were observed in the resistant S. epidermidis of 21 strains. One-point mutation was observed mainly in gyrA and parC, and a small number of mutations were observed in parE in the form of a double point mutations. When there were multiple point mutations in both gyrA and parC, the highest minimum inhibitory concentration was observed.ConclusionsThe quinolone resistance rate of S. epidermidis increased in comparison with previous studies, and resistant S. epidermidis showed mostly QRDR mutations, which were mainly found in gyrA and parC, and showed strong resistance when mutated in both genes.

Genetic Determinants of Resistance to Extended-Spectrum Cephalosporin and Fluoroquinolone in Escherichia coli Isolated from Diseased Pigs in the United States.

ABSTRACTFluoroquinolones and cephalosporins are critically important antimicrobial classes for both human and veterinary medicine. We previously found a drastic increase in enrofloxacin resistance in clinical Escherichia coli isolates collected from diseased pigs from the United States over 10 years (2006 to 2016). However, the genetic determinants responsible for this increase have yet to be determined. The aim of the present study was to identify and characterize the genetic basis of resistance against fluoroquinolones (enrofloxacin) and extended-spectrum cephalosporins (ceftiofur) in swine E. coli isolates using whole-genome sequencing (WGS). blaCMY-2 (carried by IncA/C2, IncI1, and IncI2 plasmids), blaCTX-M (carried by IncF, IncHI2, and IncN plasmids), and blaSHV-12 (carried by IncHI2 plasmids) genes were present in 87 (82.1%), 19 (17.9%), and 3 (2.83%) of the 106 ceftiofur-resistant isolates, respectively. Of the 110 enrofloxacin-resistant isolates, 90 (81.8%) had chromosomal mutations in gyrA, gyrB, parA, and parC genes. Plasmid-mediated quinolone resistance genes [qnrB77, qnrB2, qnrS1, qnrS2, and aac-(6)-lb′-cr] borne on ColE, IncQ2, IncN, IncF, and IncHI2 plasmids were present in 24 (21.8%) of the enrofloxacin-resistant isolates. Virulent IncF plasmids present in swine E. coli isolates were highly similar to epidemic plasmids identified globally. High-risk E. coli clones, such as ST744, ST457, ST131, ST69, ST10, ST73, ST410, ST12, ST127, ST167, ST58, ST88, ST617, ST23, etc., were also found in the U.S. swine population. Additionally, the colistin resistance gene (mcr-9) was present in several isolates. This study adds valuable information regarding resistance to critical antimicrobials with implications for both animal and human health.IMPORTANCE Understanding the genetic mechanisms conferring resistance is critical to design informed control and preventive measures, particularly when involving critically important antimicrobial classes such as extended-spectrum cephalosporins and fluoroquinolones. The genetic determinants of extended-spectrum cephalosporin and fluoroquinolone resistance were highly diverse, with multiple plasmids, insertion sequences, and genes playing key roles in mediating resistance in swine Escherichia coli. Plasmids assembled in this study are known to be disseminated globally in both human and animal populations and environmental samples, and E. coli in pigs might be part of a global reservoir of key antimicrobial resistance (AMR) elements. Virulent plasmids found in this study have been shown to confer fitness advantages to pathogenic E. coli strains. The presence of international, high-risk zoonotic clones provides worrisome evidence that resistance in swine isolates may have indirect public health implications, and the swine population as a reservoir for these high-risk clones should be continuously monitored.

Molecular mechanisms of fluoroquinolone resistance in Enterobacteriaceae clinical isolates in Azerbaijan, Iran

Fluoroquinolones (FQs) resistance due to their extensive clinical use has been a major problem in clinical settings that lead to limitation of treatment. This study aimed to investigate the resistance genes to FQs in Enterobacteriaceae isolates in Azerbaijan, Iran. A total of 88 Enterobacteriaceae isolated from clinical samples were collected. The presence of mutations in quinolone resistance genes were detected by the PCR and direct sequencing. The expression levels of acrB efflux pump and the ompF porin were evaluated using the Real-Time PCR. Overall, 63 isolates (71.6%) were resistant to FQs. All FQ-resistant Enterobacteriaceae had a mutation in gyrA. We found 100% and 76% mutation at Ser83Leu and Asp87Asn of gyrA, respectively. Mutations of gyrB were including Ser359Ala + Ser367Thr (1.5%) and Ser366Pro + Arg478Trp (1.5%). Also, mutations in Ser80Ile (68%) and Glu84Val (30%) were found in parC gene. The amino-acid substitutions of Ile529Leu, Ser458Ala, and Leu416Phe in parE were detected in 27%, 23.8% and 15.8% of FQ-resistant Enterobacteriaceae, respectively. Forty-eight (92%) of FQ-resistant Enterobacteriaceae were positive for plasmid-mediated quinolone resistance (PMQR) genes. The expression of acrB increased and expression level of ompF decreased by 58.7% and 64.2% FQ-resistant isolates. The high levels of resistance to FQs were observed among Enterobacteriaceae clinical isolates. We found high-level FQ resistance associated with DNA gyrase and topoisomerase IV mutations as a major mechanism, and along with PMQR genes, expression of the acrB efflux pump gene and outer membrane ompF gene in Azerbaijan, Iran.

Regional Variation of Extended-Spectrum Beta-Lactamase (ESBL)-Producing Enterobacterales, Fluoroquinolone-Resistant Salmonella enterica and Methicillin-Resistant Staphylococcus aureus Among Febrile Patients in Sub-Saharan Africa.

BackgroundAntimicrobial resistance (AMR) thwarts the curative power of drugs and is a present-time global problem. We present data on antimicrobial susceptibility and resistance determinants of bacteria the WHO has highlighted as being key antimicrobial resistance concerns in Africa, to strengthen knowledge of AMR patterns in the region.MethodsBlood, stool, and urine specimens of febrile patients, aged between ≥ 30 days and ≤ 15 years and hospitalized in Burkina Faso, Gabon, Ghana, and Tanzania were cultured from November 2013 to March 2017 (Patients > 15 years were included in Tanzania). Antimicrobial susceptibility testing was performed for all Enterobacterales and Staphylococcus aureus isolates using disk diffusion method. Extended-spectrum beta-lactamase (ESBL) production was confirmed by double-disk diffusion test and the detection of blaCTX–M, blaTEM and blaSHV. Multilocus sequence typing was conducted for ESBL-producing Escherichia coli and Klebsiella pneumoniae, ciprofloxacin-resistant Salmonella enterica and S. aureus. Ciprofloxacin-resistant Salmonella enterica were screened for plasmid-mediated resistance genes and mutations in gyrA, gyrB, parC, and parE. S. aureus isolates were tested for the presence of mecA and Panton-Valentine Leukocidin (PVL) and further genotyped by spa typing.ResultsAmong 4,052 specimens from 3,012 patients, 219 cultures were positive of which 88.1% (n = 193) were Enterobacterales and 7.3% (n = 16) S. aureus. The prevalence of ESBL-producing Enterobacterales (all CTX-M15 genotype) was 45.2% (14/31; 95% CI: 27.3, 64.0) in Burkina Faso, 25.8% (8/31; 95% CI: 11.9, 44.6) in Gabon, 15.1% (18/119; 95% CI: 9.2, 22.8) in Ghana and 0.0% (0/12; 95% CI: 0.0, 26.5) in Tanzania. ESBL positive non-typhoid Salmonella (n = 3) were detected in Burkina Faso only and methicillin-resistant S. aureus (n = 2) were detected in Ghana only. While sequence type (ST)131 predominated among ESBL E. coli (39.1%;9/23), STs among ESBL K. pneumoniae were highly heterogenous. Ciprofloxacin resistant nt Salmonella were commonest in Burkina Faso (50.0%; 6/12) and all harbored qnrB genes. PVL were found in 81.3% S. aureus.ConclusionOur findings reveal a distinct susceptibility pattern across the various study regions in Africa, with notably high rates of ESBL-producing Enterobacterales and ciprofloxacin-resistant nt Salmonella in Burkina Faso. This highlights the need for local AMR surveillance and reporting of resistances to support appropriate action.

QUANTITATIVE ANALYSIS OF GYRA AND GYRB KNOWN MUTATIONS IN DRUG-RESISTANT MYCOBACTERIUM TUBERCULOSIS STRAINS TREATED WITH OFLOXACIN

Objective: The aim of the study is to measure the minimum inhibitory concentrations (MICs) of ofloxacin antibiotic from gyrA and gyrB mutations present in fluoroquinolones (FQs) resistant strains of Mycobacterium tuberculosis (MTB) and to further concentrate the potential association between gene mutations and phenotypic resistance based on their MICs.
 Methods: Different levels of ofloxacin MICs levels were detected in 31 archived multi drug-resistant MTB isolates showing gyrA mutations in codon at A90V, S91P, D94A, D94N/Y, D94 G, and D94H and two gyrB probes (N538D and E540V). The MIC determinations were made using the 1% proportion method.
 Results: Genotypic assay detected 32 mutations in the gyrA (n=29) and gyrB (n=3) genes among the 31 FQs resistant isolates. Most frequently seen in gyrA mutations at codon D94G (n=16; 50%), these mutations had a clearly elevated MIC level from 2 to 16 μg/ml, that was phenotypically resistant. The A90V mutation region consistently exhibited the lowest levels of ofloxacin resistance, with three out of eight (37.50%) of these isolates had a MIC of <2 μg/ml. In addition, a further strain of S91P mutations for MIC was determined to be less than the critical concentration (CC). These low levels of resistance have been detected in a phenotypic manner which is noticeable in the study. Furthermore, fewer mutations in codons at D94A, D94N/Y were identified. Three wild-type absent isolates from gyrB QRDR were identified and the MIC of those strains for ofloxacin was lower than the critical cutoff value.
 Conclusion: Based on the results, it is shown that different resistance mutations were associated with different levels of MICs and the current concentration for MGIT will be lowered from 2 μg/ml to 1 μg/ml for the ofloxacin drug.

Phenotypic and genotypic quinolone resistance in Escherichia coli underlining GyrA83/87 mutations as a target to detect ciprofloxacin resistance.

Quinolone resistance (QR) is one component of the MDR emerging in Escherichia coli and is of particular concern given the widespread use of fluoroquinolones. To characterize the QR phenotypes and genotypes in E. coli responsible for bloodstream infections and to propose molecular determinants that could be targeted to predict ciprofloxacin resistance. E. coli isolates from blood cultures in three French hospitals were studied for quinolone MICs and characterization of genotypic QR determinants (QRg). Among 507 isolates tested for MICs, 148 (29.2%) were resistant to quinolones based on EUCAST breakpoints and 143 (28.2%) harboured at least one QRg. QRg were mainly mutations in the QRDR (138 isolates, 27.2%), with 55.8% of these isolates carrying at least three QRDR mutations. gyrA mutations predominated (92.8%) followed by parC (61.6%), parE (32.6%) and gyrB (1.4%) mutations. Only 4.7% of the isolates harboured a plasmid-mediated quinolone resistance (PMQR) gene: aac(6')-Ib-cr (60.0%) or qnr (qnrS, qnrB) (32.0%). For the first time in France, we reported the qepA4 allele of the plasmid-encoded efflux pump QepA. Only five isolates carried PMQR without a QRDR mutation. The positive predictive value (PPV) for ciprofloxacin resistance was 100% for any QRg and 99.2% for gyrA mutations specifically. QR observed in E. coli isolates involved in bloodstream infections is still mainly due to QRDR mutations, especially at codons GyrA83/87, which could be used as a molecular target to rapidly detect resistance.

Genomic profiling of antimicrobial resistance genes in clinical isolates of Salmonella Typhi from patients infected with Typhoid fever in India

The development of multidrug resistance in Salmonella enterica serovar Typhi currently forms a major roadblock for the treatment of enteric fever. This poses a major health problem in endemic regions and extends to travellers returning from developing countries. The appearance of fluoroquinolone non-susceptible strains has resulted in use of ceftriaxone as drug of choice with azithromycin being recommended for uncomplicated cases of typhoid fever. A recent sporadic instance of decreased susceptibility to the latest drug regime has necessitated a detailed analysis of antimicrobial resistance genes and possible relationships with their phenotypes to facilitate selection of future treatment regimes. Whole genome sequencing (WGS) was conducted for 133 clinical isolates from typhoid patients. Sequence output files were processed for pan-genome analysis and prediction of antimicrobial resistance genes. The WGS analyses disclosed the existence of fluoroquinolone resistance conferring mutations in gyrA, gyrB, parC and parE genes of all strains. Acquired resistance determining mechanisms observed included catA1 genes for chloramphenicol resistance, dfrA7, dfrA15, sul1 and sul2 for trimethoprim-sulfamethoxazole and blaTEM-116/blaTEM-1B genes for amoxicillin. No resistance determinants were found for ceftriaxone and cefixime. The genotypes were further correlated with their respective phenotypes for chloramphenicol, ampicillin, co-trimoxazole, ciprofloxacin and ceftriaxone. A high correlation was observed between genotypes and phenotypes in isolates of S. Typhi. The pan-genome analysis revealed that core genes were enriched in metabolic functions and accessory genes were majorly implicated in pathogenesis and antimicrobial resistance. The pan-genome of S. Typhi appears to be closed (Bpan = 0.09) as analysed by Heap’s law. Simpson’s diversity index of 0.51 showed a lower level of genetic diversity among isolates of S. Typhi. Overall, this study augments the present knowledge that WGS can help predict resistance genotypes and eventual correlation with phenotypes, enabling the chance to spot AMR determinants for fast diagnosis and prioritize antibiotic use directly from sequence.

Fluoroquinolone resistance and mutational profile of gyrA in pulmonary MDR tuberculosis patients

BackgroundFluoroquinolones (FQs) are potential drugs that inhibit DNA synthesis and are used in the treatment of multidrug-resistant tuberculosis (TB) and short-term anti-TB regimens. In recent years, a high proportion of FQ resistance has been observed in Mycobacterium tuberculosis isolates. The development of FQ resistance in multidrug-resistant TB negatively impacts patient treatment outcome and is a serious threat to control of TB.MethodsThe study included a total of 562 samples from patients with pulmonary TB that had been on anti-tuberculosis therapy. MTBDRsl assays were performed for the molecular detection of mutations. Sequence analysis was performed for the characterization and mutational profiling of FQ-resistant isolates.ResultsFQ resistance was observed in 104 samples (18.5%), most of which were previously treated and treatment failure cases. A total of 102 isolates had mutations in DNA gyrase subunit A (gyrA), while mutations in gyrB were observed in only two isolates. Mutational analysis revealed that the mutations mostly alter codons 94 (replacing aspartic acid with glycine, D94G) and 90 (replacing alanine with valine, A90V). In MDR and treatment failure cases, resistance to FQs was most commonly associated with the D94G mutation. In contract, a high proportion of A90V mutations were observed in isolates that were newly diagnosed.ConclusionThe findings suggest that genotypic assays for FQ resistance should be carried out at the time of initial diagnosis, before starting treatment, in order to rule out mutations that impact the potential use of FQs in treatment and to control drug resistance.

Discovery of seven novel mutations of gyrB, parC and parE in Salmonella Typhi and Paratyphi strains from Jiangsu Province of China

Objective: To investigate the prevalence of Salmonella Typhi and Paratyphi resistance to quinolones and characterize the underlying mechanism in Jiangsu Province of China. Methods: Antimicrobial susceptibility testing was performed using Kirby-Bauer disc diffusion system. Quinolone resistance-determining region (QRDR), plasmid-mediated quinolone resistance (PMQR) determinant genes were detected by PCR and sequencing. Results: Out of 239 Salmonella isolates, 164 were S. Typhi and 75 were S. Paratyphi. 128 (53.6%) Salmonella isolates were resistant to nalidixic acid; 11 (4.6%) isolates to ciprofloxacin and 66 (27.6%) isolates were intermediate to ciprofloxacin. QRDR were present in 69 S. Typhi isolates, among which mutation at codon 83 (n = 45) and 133 (n = 61) predominated. In S. Paratyphi, the most common mutations were detected in gyrA at codon 83(n = 24) and parC: T57S (n = 8). Seven mutations were first reported in Salmonella isolates including gyrB: S426G, parC: D79G and parE: [S498T, E543K, V560G, I444S, Y434S]. PMQR genes including qnrD1, qnrA1, qnrB4, aac (6′)-Ib-cr4 and qnrS1 were detected in 1, 2, 3, 7 and 9 isolates, relatively. Conclusions: High resistance to quinolones in Salmonella remains a serious problem in Jiangsu, China. The presence of the novel mutations increases the complexity of quinolone-resistant genotypes and poses a threat to public health. Subject terms: Salmonella Typhi, Salmonella Paratyphi, antimicrobial resistance, QRDR, PMQR.

The tcdA-negative and tcdB-positive Clostridium difficile ST81 clone exhibits a high level of resistance to fluoroquinolones: a multi-centre study in Beijing, China

Clostridium difficile infection (CDI) is the leading cause of antibiotic-associated diarrhoea worldwide. In order to gain a better understanding about the molecular epidemiology of C. difficile in Beijing, China, molecular typing, antimicrobial susceptibility testing and drug resistance gene sequencing were performed on 174 strains of C. difficile collected from four large tertiary hospitals in Beijing. In total, 31 sequence types (STs) were identified among the 174 strains. ST81 was found to be the most prevalent (26.4%, 46/174), followed by ST2 (16.7%, 29/174) and ST54 (9.8%, 17/174). All isolates were susceptible to metronidazole and vancomycin. The test strains displayed resistance rates of 97.1%, 44.3% and 44.3% for ciprofloxacin, levofloxacin and moxifloxacin, respectively. ST81 isolates displayed a drug resistance rate of 97.8% for levofloxacin and moxifloxacin, which was significantly higher than ST2 (0%), ST54 (17.6%) and ST42 (0%) isolates (P<0.05). An amino acid mutation (T82I) was identified in GyrA, and the total mutation rate of the C. difficile strains was 40.8% (71/174). The mutation rate of ST81 isolates was 95.7% (44/46). Three amino acid mutations (D426N, S366A and D426V) were identified in GyrB, and the total mutation rate of GyrB was 39.1%. A double-site mutation in GyrB (S366A+D426V) was identified in all ST81 (n=46) isolates. In conclusion, the C. difficile ST81 clone showed a high level of resistance to fluoroquinolones in Beijing, highlighting the need for nationwide surveillance of CDI.

Frequency of first and second-line drug resistance-associated mutations among resistant Mycobacterium tuberculosis clinical isolates from São Paulo, Brazil.

BACKGROUND Tuberculosis (TB) is an infectious disease caused by the bacterium Mycobacterium tuberculosis, and the number of new cases of multidrug resistant TB (MDR-TB), pre extensively drug-resistant TB (pre-XDR-TB) and extensively drug-resistant TB (XDR-TB) has increased considerably worldwide.OBJECTIVES Herein, using 156 M. tuberculosis isolates from 106 patients previously classified as MDR or pre-XDR or XDR isolates, we investigated the genetic mutation profiles associated with phenotypic resistances in patients with MDR-TB, pre-XDR-TB and XDR-TB, treatment outcomes and resistance evolution.METHODS Molecular analyses were performed by partial sequencing of the rpoB, katG, gyrA, gyrB, rrs genes and analysis of the fabG-inhA promoter region. Clinical, epidemiologic and demographic data were obtained from the TB Notification database system of São Paulo (TB-WEB) and the Information System for Special Tuberculosis Treatments (SITE-TB).FINDINGS Drug resistance was attributed to previously known mutations and a novel Asp449Val mutation in gyrB was observed in four isolates from the same patient. Ten patients had more than one isolate evaluated and eight of these patients displayed resistance progression.MAIN CONCLUSIONS The present study is the first to report the frequency of mutations related to second-line drug resistance in MDR-TB, pre-XDR-TB and XDR-TB isolates. The results could lead to the improvement of available technologies for the rapid detection of drug resistant TB.

DNA Gyrase and Topoisomerase IV Mutations and their effect on Quinolones Resistant Proteus mirabilis among UTIs Patients.

Objective:This study aimed to highlight the importance of mutations within Proteus mirabilis genome that are related to fluoroquinolone resistance.Methods:This is a cross sectional study performed in different teaching hospitals in Khartoum State from June 2016 to May 2017. A total of (120) P mirabilis isolates from patients with symptoms of UTIs attending different hospitals in Khartoum State were examined. First, modified Kurby Bauer method was performed for phenotypical detection of resistant isolates. Then polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) followed by sequencing were applied for detection of mutations in GyrA, GyrB, ParC and ParE genes of isolates.Results:P. mirabilis showed 30% resistance to ciprofloxacin. All samples revealed mutation at (serine 83) of GyrA and (serine 84) of ParC by Hinf1 restriction endonuclease digestion. Sequencing was performed for 12 samples. For each gene, two resistant and one susceptible strains were randomly selected. The mutations associated with ciprofloxacin resistant P. mirabilis were as follows; (1/3) GyrA (Ser 83 to Ile) and (2/3) ParC (Ser 81 to Ile). Also it revealed silent mutations at codons of GyrB 474 leucine (3/3), 585 valine (2/3), 612 histidine (1/3) and 639 asparagine (1/3) and ParE 469 isoleucine (2/3), 531 aspartic (2/3) and 533 glycine (1/3).Conclusions:Ciprofloxacin resistance in P. mirabilis could be monitored through detection of mutations within DNA gyrase (encoded by gyrA and gyrB) and topoisomerase IV (encoded by parC and parE).

Characterization of Mechanisms Lowering Susceptibility to Flumequine among Bacteria Isolated from Chilean Salmonid Farms.

Despite their great importance for human therapy, quinolones are still used in Chilean salmon farming, with flumequine and oxolinic acid currently approved for use in this industry. The aim of this study was to improve our knowledge of the mechanisms conferring low susceptibility or resistance to quinolones among bacteria recovered from Chilean salmon farms. Sixty-five isolates exhibiting resistance, reduced susceptibility, or susceptibility to flumequine recovered from salmon farms were identified by their 16S rRNA genes, detecting a high predominance of species belonging to the Pseudomonas genus (52%). The minimum inhibitory concentrations (MIC) of flumequine in the absence and presence of the efflux pump inhibitor (EPI) Phe-Arg-β-naphthylamide and resistance patterns of isolates were determined by a microdilution broth and disk diffusion assays, respectively, observing MIC values ranging from 0.25 to >64 µg/mL and a high level of multi-resistance (96%), mostly showing resistance to florfenicol and oxytetracycline. Furthermore, mechanisms conferring low susceptibility to quinolones mediated by efflux pump activity, quinolone target mutations, or horizontally acquired resistance genes (qepA, oqxA, aac(6′)-lb-cr, qnr) were investigated. Among isolates exhibiting resistance to flumequine (≥16 µg/mL), the occurrence of chromosomal mutations in target protein GyrA appears to be unusual (three out of 15), contrasting with the high incidence of mutations in GyrB (14 out of 17). Bacterial isolates showing resistance or reduced susceptibility to quinolones mediated by efflux pumps appear to be highly prevalent (49 isolates, 75%), thus suggesting a major role of intrinsic resistance mediated by active efflux.

The mutational landscape of quinolone resistance in Escherichia coli.

The evolution of antibiotic resistance is influenced by a variety of factors, including the availability of resistance mutations, and the pleiotropic effects of such mutations. Here, we isolate and characterize chromosomal quinolone resistance mutations in E. coli, in order to gain a systematic understanding of the rate and consequences of resistance to this important class of drugs. We isolated over fifty spontaneous resistance mutants on nalidixic acid, ciprofloxacin, and levofloxacin. This set of mutants includes known resistance mutations in gyrA, gyrB, and marR, as well as two novel gyrB mutations. We find that, for most mutations, resistance tends to be higher to nalidixic acid than relative to the other two drugs. Resistance mutations had deleterious impacts on one or more growth parameters, suggesting that quinolone resistance mutations are generally costly. Our findings suggest that the prevalence of specific gyrA alleles amongst clinical isolates are driven by high levels of resistance, at no more cost than other resistance alleles.

In vitro antimicrobial combination testing of and evolution of resistance to the first-in-class spiropyrimidinetrione zoliflodacin combined with six therapeutically relevant antimicrobials for Neisseria gonorrhoeae.

Resistance in Neisseria gonorrhoeae to all gonorrhoea therapeutic antimicrobials has emerged. Novel therapeutic antimicrobials are imperative and the first-in-class spiropyrimidinetrione zoliflodacin appears promising. Zoliflodacin could be introduced in dual antimicrobial therapies to prevent the emergence and/or spread of resistance. We investigated the in vitro activity of and selection of resistance to zoliflodacin alone and in combination with six gonorrhoea therapeutic antimicrobials against N. gonorrhoeae. The international gonococcal reference strains WHO F (WT) and WHO O, WHO V and WHO X (strains with different AMR profiles) were examined. Zoliflodacin was evaluated alone or combined with ceftriaxone, cefixime, spectinomycin, gentamicin, tetracycline, cethromycin or sitafloxacin in chequerboard assays, time-kill curve analysis and selection-of-resistance studies. Zoliflodacin alone or in combination with all six antimicrobials showed rapid growth inhibition against all examined strains. The time-kill curve analysis indicated that tetracycline or cethromycin combined with zoliflodacin can significantly decrease the zoliflodacin kill rate in vitro. The frequency of selected zoliflodacin-resistance mutations was low when evaluated as a single agent and further reduced for all antimicrobial combinations. All resistant mutants contained the GyrB mutations D429N, K450T or K450N, resulting in zoliflodacin MICs of 0.5-4 mg/L. Zoliflodacin, alone or in combination with sexually transmitted infection therapeutic antimicrobials, rapidly kills gonococci with infrequent resistance emergence. Zoliflodacin remains promising for gonorrhoea oral monotherapy and as part of dual antimicrobial therapy with low resistance emergence potential. A Phase III trial evaluating efficacy and safety of zoliflodacin for uncomplicated gonorrhoea treatment is planned in 2019.

Mutation and Transmission Profiles of Second-Line Drug Resistance in Clinical Isolates of Drug-Resistant Mycobacterium tuberculosis From Hebei Province, China.

The emergence of drug-resistant tuberculosis (TB) is involved in ineffective treatment of TB, especially multidrug resistant/extensively resistant TB (MDR/XDR-TB), leading to acquired resistance and transmission of drug-resistant strains. Second-line drugs (SLD), including both fluoroquinolones and injectable drugs, were commonly proved to be the effective drugs for treatment of drug-resistant TB. The purpose of this study was to investigate the prevalence of SLD-resistant strains and its specific mutations in drug-resistant Mycobacterium tuberculosis clinical isolates, and to acknowledge the transmission pattern of SLD resistance strains in Hebei. The genes gyrA, gyrB, rrs, eis promoter and tlyA of 257 drug-resistant clinical isolates were sequenced to identify mutations that could be responsible for resistance against fluoroquinolones and second-line injectable drugs. Each isolate was genotyped by Spoligotyping and 15-loci MIRU-VNTR. Our results indicated that 48.2% isolates were resistant to at least one of five SLD. Of them, 37.7% isolates were resistant to fluoroquinolones and 24.5% isolates were resistant to second-line injectable drugs. Mutations in genes gyrA, gyrB, rrs, eis promoter and tlyA were detected in 73 (75.3%), 7 (7.2%), 24 (38.1%), 5 (7.9%), and 3 (4.8%) isolates, respectively. The most prevalent mutations were the D94G (23.7%) in gyrA gene and the A1401G (33.3%) in rrs gene. A combination of gyrA, rrs and eis promoter can act as a valuable predicator for predicting XDR phenotype. These results highlight the development of rapid diagnosis are the effective manners for the control of SLD-TB or XDR-TB.