Fluoroquinolone resistance among fecal extended spectrum ßeta lactamases positive Enterobacterales isolates from children in Dar es Salaam, Tanzania.

BACKGROUND: Fluoroquinolones have been, and continue to be, routinely used for treatment of many bacterial infections. In recent years, most parts of the world have reported an increasing trend of fluoroquinolone resistant (FQR) Gram-negative bacteria. METHODS: A cross-sectional study was conducted between March 2017 and July 2018 among children admitted due to fever to referral hospitals in Dar es Salaam, Tanzania. Rectal swabs were used to screen for carriage of extended-spectrum ß-lactamase-producing Enterobacterales (ESBL-PE). ESBL-PE isolates were tested for quinolone resistance by disk diffusion method. Randomly selected fluroquinolone resistant isolates were characterized by using whole genome sequencing. RESULTS: A total of 142 ESBL-PE archived isolates were tested for fluoroquinolone resistance. Overall phenotypic resistance to ciprofloxacin, levofloxacin and moxifloxacin was found in 68% (97/142). The highest resistance rate was seen among Citrobacter spp. (100%, 5/5), followed by Klebsiella. pneumoniae (76.1%; 35/46), Escherichia coli (65.6%; 42/64) and Enterobacter spp. (31.9%; 15/47). Whole genome sequencing (WGS) was performed on 42 fluoroquinolone resistant-ESBL producing isolates and revealed that 38/42; or 90.5%, of the isolates carried one or more plasmid mediated quinolone resistance (PMQR) genes. The most frequent PMQR genes were aac(6')-lb-cr (74%; 31/42), followed by qnrB1 (40%; 17/42), oqx, qnrB6 and qnS1. Chromosomal mutations in gyrA, parC and parE were detected among 19/42 isolates, and all were in E. coli. Most of the E. coli isolates (17/20) had high MIC values of > 32 µg/ml for fluoroquinolones. In these strains, multiple chromosomal mutations were detected, and all except three strains had additional PMQR genes. Sequence types, ST131 and ST617 predominated among E. coli isolates, while ST607 was more common out of 12 sequence types detected among the K. pneumoniae. Fluoroquinolone resistance genes were mostly associated with the IncF plasmids. CONCLUSION: The ESBL-PE isolates showed high rates of phenotypic resistance towards fluoroquinolones likely mediated by both chromosomal mutations and PMQR genes. Chromosomal mutations with or without the presence of PMQR were associated with high MIC values in these bacteria strains. We also found a diversity of PMQR genes, sequence types, virulence genes, and plasmid located antimicrobial resistance (AMR) genes towards other antimicrobial agents.

Relapse in patients with schizophrenia and amisulpride-induced hyperprolactinemia or olanzapine-induced metabolic disturbance after switching to other antipsychotics.

Hyperprolactinemia and metabolic disturbance are common side effects of antipsychotics that cause intolerance. Despite its potential influence on relapse, there are no established guidelines for antipsychotic switching. This naturalistic study explored the association between antipsychotic switching, baseline clinical status, metabolic changes, and relapse in patients with schizophrenia. In total, 177 patients with amisulpride-induced hyperprolactinemia and 274 with olanzapine-induced metabolic disturbance were enrolled. Relapse was determined by assessing changes in Positive and Negative Syndrome Scale (PANSS) total scores from baseline to 6 months (increased over 20% or 10% reaching 70). Metabolic indices were measured at baseline and 3 months. Patients with baseline PANSS >60 were more likely to relapse. Further, patients switching to aripiprazole had a higher risk of relapse regardless of their original medication. Participants who originally used amisulpride had reduced prolactin levels following medication change, while switching to olanzapine caused increased weight and blood glucose levels. In patients originally using olanzapine, only switching to aripiprazole reduced insulin resistance. Adverse effects on weight and lipid metabolism were observed in patients who switched to risperidone, while amisulpride improved lipid profiles. Changing schizophrenia treatment requires careful consideration of multiple variables, particularly the choice of substituted drug and the patient's baseline symptoms.

Hydrogen Bond Assisted Three-Component Tandem Reactions to Access N-Alkyl-4-Quinolones.

Hydrogen-bonding catalytic reactions have gained great interest. Herein, a hydrogen-bond-assisted three-component tandem reaction for the efficient synthesis of N-alkyl-4-quinolones is described. This novel strategy features the first proof of polyphosphate ester (PPE) as a dual hydrogen-bonding catalyst and the use of readily available starting materials for the preparation of N-alkyl-4-quinolones. The method provides a diversity of N-alkyl-4-quinolones in moderate to good yields. The compound 4h demonstrated good neuroprotective activity against N-methyl-ᴅ-aspartate (NMDA)-induced excitotoxicity in PC12 cells.

[Occurrence and resistance of bacteria isolated from the rabbit nares - a retrospective evaluation]. / Vorkommen und Resistenzen von Bakterien in Proben aus Kaninchennasen ­ eine retrospektive Auswertung.

OBJECTIVE: Rabbit snuffles is one of the most common challenges in veterinary practice. The aim of the present study was to evaluate nasal samples of rabbits submitted between 2015-2019, with regard to bacterial distribution and antimicrobial resistance. MATERIAL AND METHODS: Each sample was plated on four different agar plates and enriched in a non-selective broth. Isolates were identified by MALDI Biotyper® (MBT) (Bruker Daltonik GmbH, Bremen, Germany) and antimicrobial susceptibility was performed by broth microdilution method in accordance with the Clinical and Laboratory Standards Institute (CLSI, Wayne, PA, USA). RESULTS: A total of 1261 samples were evaluated. Among the samples that tested positive (n=941), one bacterial species was detected in 79.1% of the cases, and more than one bacterial species (mixed culture) was found in 20.9% of the cases. A total of 150 species from 14 families were identified. Isolates belonging to the family Pasteurellaceae were identified most frequently, followed by Enterobacteriaceae, Pseudomonadaceae and Staphylococcaceae.A total of 467 antibiograms of the most common pathogens with possible clinical relevance (Pasteurella multocida [14.6%], Pasteurella species [10.0%], Staphylococcus aureus [5.9%], Pseudomonas aeruginosa [5.4%] and Bordetella bronchiseptica [4.8%]) were evaluated. Quinolones showed the highest efficacy and clindamycin the lowest. Furthermore, among S. aureus, MRSA were most frequently detected in 2016 reaching 23.1% of cases. CONCLUSIONS AND CLINICAL RELEVANCE: Since the causal bacteria for rabbit snuffles are mostly found in the deeper areas of the nose and the nasal vestibule is often contaminated with ubiquitous and coliform bacteria, it would make sense to take samples from the depth of the nasal cavity, ideally via nasal lavage. Due to the demonstrated pathogen diversity and long-term therapy associated with the disease, bacterial culture and sensitivity testing is recommended as part of the management. In the absence of an antibiogram, enrofloxacin is the drug of first choice due to its favorable resistance pattern and tolerability. However, since quinolones are considered as "critically important" antibiotics, their use should be limited to a minimum.

Design, Synthesis, and Pharmacology of New Triazole-Containing Quinolinones as CNS Active Agents.

Epilepsy and major depressive disorder are the two of the most common central nervous system (CNS) diseases. Clinicians and patients call for new antidepressants, antiseizure medicines, and in particular drugs for depression and epilepsy comorbidities. In this work, a dozen new triazole-quinolinones were designed, synthesized, and investigated as CNS active agents. All compounds reduced the immobility time significantly during the forced swim test (FST) in mice at the dosage of 50 mg/kg. Compounds 3f-3j gave superior performance over fluoxetine in the FST with more reductions of the immobility time. Compound 3g also reduced immobility time significantly in a tail suspension test (TST) at the dosage of 50 mg/kg, though its anti-immobility activity was inferior to that of fluoxetine. An open field test was carried out and it eliminated the false-positive possibility of 3g in the FST and TST, which complementarily supported the antidepressant activity of 3g. We also found that almost all compounds except 3k exhibited antiseizure activity in the maximal electroshock seizure (MES) model at 100 or 300 mg/kg. Compounds 3c, 3f, and 3g displayed the ED50 of 63.4, 78.9, and 84.9 mg/kg, and TD50 of 264.1, 253.5, and 439.9 mg/kg, respectively. ELISA assays proved that the mechanism for the antiseizure and antidepressant activities of compound 3g was via affecting the concentration of GABA in mice brain. The molecular docking study showed a good interaction between 3g and the amino acid residue of the GABAA receptor. Excellent drug-like properties and pharmacokinetic properties of compound 3a-l were also predicted by Discovery Studio. These findings provided a new skeleton to develop agents for the treatment of epilepsy and depression comorbidities.

MLST genotypes and quinolone resistance profiles of Campylobacter jejuni isolates from various sources in Turkey.

This study was conducted to determine the overall genetic diversity, as well as prevalence and mechanisms of resistance to quinolone antibiotics of 178 Campylobacter jejuni isolated from humans, cattle, dogs, and chickens in Turkey. Multilocus sequence typing (MLST) and E-test were performed for genotyping and antimicrobial susceptibility testing, respectively. Mismatch Amplification Mutation Assay, Polymerase Chain Reaction (MAMA-PCR) was used to detect point mutations associated with quinolone resistance. Of the 178 isolates tested, 151 were included in 21 clonal complexes (CCs); the remaining 27 isolates did not belong to any existing CCs. CC21, CC353, CC206, and CC257 were the predominant clones, representing 38 % of all C. jejuni isolates tested. The isolates were assigned to 78 different sequence types (STs), three of which were novel (ST 8082, ST 8083, and ST 8084). Resistance to quinolones was found in 73 (41 %) of the isolates (42.85 %, 2.85 %, 20.58 %, and 43.75 % in human, cattle, dog, and chicken isolates, respectively). All of the resistant isolates had Thr-86-Ile mutation in the gyrA gene. The highest Sorensen coefficient index was detected for human/chicken meat and human/dog C. jejuni isolates (Ss = 0.71), suggesting a strong link between the isolates from respective sources. The Simpson diversity index of C. jejuni isolates analyzed was detected between 0.92 and 0.98. The study provides detailed information on the quinolone resistance and MLST-based genetic relatedness of C. jejuni isolates from humans, cattle, dog, and broiler meat in Turkey for the first time, enabling a better understanding of the transmission pathways of C. jejuni in this country. Our results suggest that broiler meat and dogs may be the most important sources of human campylobacteriosis in Turkey.

Occurrence, source apportionment and ecological risk assessment of thirty antibiotics in farmland system.

Antibiotics are widely used in medical care, livestock production, and aquaculture. However, antibiotic pollution has attracted increasing global concerns due to their ecological risks after entering into environmental ecosystem via animal excretion, effulent from industrial and domestic sewage treatment facilities. In this study, 30 antibiotics were investigated in soils and irrigation rivers using ultra-performance liquid chromatography-triple quadrupole tandem mass spectrometer. This study evaluated the occurrence, source apportionment, and ecological risks of these target compounds in soils and irrigation rivers (i.e., sediments and water) of farmland system by using principal component analysis-multivariate linear regression (PCA-MLR) and risk quotients (RQ). The concentration range of antibiotics in soils, sediments, and water was 0.38-689.58 ng/g, 81.99-658.00 ng/g, and 134.45-1547.06 ng/L, respectively. In soils, the most abundant antibiotics were quinolones and antifungals with an average concentration of 30.00 ng/g and 7.69 ng/g, respectively, contributing to 40% of total antibiotics. Macrolides were the most frequently detected antibiotics in soils with an average concentration of 4.94 ng/g. In irrigation rivers, quinolones and tetracyclines, the most abundant antibiotics, accounted for 78% and 65% of antibiotics in water and sediments, respectively. Higher antibiotic contamination of irrigation water was primarily distributed in highly populated urban areas, while increasing antibiotic contamination of sediments and soils was particularly observed in rural areas. PCA-MLR analysis indicated that antibiotic contamination in soils was mainly ascribed to the irrigation of sewage-receiving water body and manure application of livestock and poultry farming, which cumulatively contributed to 76% of antibiotics. According to RQ assessment, quinolones in irrigation rivers posed high risk to algae and daphnia, contributing 85% and 72% to the mixture risk, respectively. In soils, macrolides, quinolones and sulfonamides were responsible for more than 90% to the mixture risk of antibiotics. Ultimately, these findings can improve our fundamental knowledge on contamination characteristics and source pathways towards risk management of antibiotics in farmland system.

Design, synthesis and biological evaluations of quinolone amides against African trypanosomiasis with improved solubility.

The human African trypanosomiasis is a devastating parasitic infection, which is caused by the protozoan Trypanosoma brucei and transmitted by the bite of the tsetse fly. An untreated infection usually results in death and only few drugs with significant drawbacks are currently available for treatment. Previous investigations revealed the quinolone amide MB007 as a lead compound with an excellent selectivity for T. b. brucei. Here, new quinolone amides were synthesized for deeper insights into the structure-activity relationship. Furthermore, the aqueous solubility of the compounds was analyzed, as the poor solubility of previous quinolone amides impeded in vivo studies for target identification. The biological evaluation led to the new lead structure 9f, which exhibits a promising in vitro activity against T. b. brucei (IC50 = 22 nM) and showed no cytotoxicity against macrophages. Moreover, compounds 10b and 10c were discovered, which possessed an improved solubility combined with a decent selectivity.

Bestatin analogs-4-quinolinone hybrids as antileishmanial hits: Design, repurposing rational, synthesis, in vitro and in silico studies.

Amongst different forms of leishmaniasis, visceral leishmaniasis caused by L. donovani is highly mortal. Identification of new hit compounds might afford new starting points to develop novel therapeutics. In this lieu, a rationally designed small library of bestatin analogs-4-quinolone hybrids were prepared and evaluated. Analysis of SAR unveiled distinct profiles for hybrids type 1 and type 2, which might arise from their different molecular targets. Amongst type 1 bestatin analog-4-quinolone hybrids, hybrid 1e was identified as potential hit inhibiting growth of L. donovani promastigotes by 91 and 53% at 50 and 25 µM concentrations, respectively. Meanwhile, hybrid 2j was identified amongst type 2 bestatin analog-4-quinolone hybrids as potential hit compound inhibiting growth of L. donovani promastigotes by 50 and 38% at 50 and 25 µM concentrations, respectively. Preliminary safety evaluation of the promising hit compounds showed that they are 50-100 folds safer against human derived monocytic THP-1 cells relative to the drug erufosine. In silico study was conducted to predict the possible binding of hybrid 1e with methionine aminopeptidases 1 and 2 of L. donovani. Molecular dynamic simulations verified the predicted binding modes and provide more in depth understanding of the impact of hybrid 1e on LdMetAP-1 and LdMetAP-2.

7-N-Substituted-3-oxadiazole Quinolones with Potent Antimalarial Activity Target the Cytochrome bc1 Complex.

The development of new antimalarials is required because of the threat of resistance to current antimalarial therapies. To discover new antimalarial chemotypes, we screened the Janssen Jumpstarter library against the P. falciparum asexual parasite and identified the 7-N-substituted-3-oxadiazole quinolone hit class. We established the structure-activity relationship and optimized the antimalarial potency. The optimized analog WJM228 (17) showed robust metabolic stability in vitro, although the aqueous solubility was limited. Forward genetic resistance studies uncovered that WJM228 targets the Qo site of cytochrome b (cyt b), an important component of the mitochondrial electron transport chain (ETC) that is essential for pyrimidine biosynthesis and an established antimalarial target. Profiling against drug-resistant parasites confirmed that WJM228 confers resistance to the Qo site but not Qi site mutations, and in a biosensor assay, it was shown to impact the ETC via inhibition of cyt b. Consistent with other cyt b targeted antimalarials, WJM228 prevented pre-erythrocytic parasite and male gamete development and reduced asexual parasitemia in a P. berghei mouse model of malaria. Correcting the limited aqueous solubility and the high susceptibility to cyt b Qo site resistant parasites found in the clinic will be major obstacles in the future development of the 3-oxadiazole quinolone antimalarial class.

Synergistic Effect of SOS Response and GATC Methylome Suppression on Antibiotic Stress Survival in Escherichia coli.

The suppression of the SOS response has been shown to enhance the in vitro activity of quinolones. Furthermore, Dam-dependent base methylation has an impact on susceptibility to other antimicrobials affecting DNA synthesis. Here, we investigated the interplay between these two processes, alone and in combination, in terms of antimicrobial activity. A genetic strategy was used employing single- and double-gene mutants for the SOS response (recA gene) and the Dam methylation system (dam gene) in isogenic models of Escherichia coli both susceptible and resistant to quinolones. Regarding the bacteriostatic activity of quinolones, a synergistic sensitization effect was observed when the Dam methylation system and the recA gene were suppressed. In terms of growth, after 24 h in the presence of quinolones, the Δdam ΔrecA double mutant showed no growth or delayed growth compared to the control strain. In bactericidal terms, spot tests showed that the Δdam ΔrecA double mutant was more sensitive than the ΔrecA single mutant (about 10- to 102-fold) and the wild type (about 103- to 104-fold) in both susceptible and resistant genetic backgrounds. Differences between the wild type and the Δdam ΔrecA double mutant were confirmed by time-kill assays. The suppression of both systems, in a strain with chromosomal mechanisms of quinolone resistance, prevents the evolution of resistance. This genetic and microbiological approach demonstrated the enhanced sensitization of E. coli to quinolones by dual targeting of the recA (SOS response) and Dam methylation system genes, even in a resistant strain model.

Discovery of New Quinolone-Based Diarylamides as Potent B-RAFV600E/C-RAF Kinase Inhibitors Endowed with Promising In Vitro Anticancer Activity.

The emergence of cancer resistance to targeted therapy represents a significant challenge in cancer treatment. Therefore, identifying new anticancer candidates, particularly those addressing oncogenic mutants, is an urgent medical demand. A campaign of structural modifications has been conducted to further optimize our previously reported 2-anilinoquinoline-diarylamides conjugate VII as a B-RAFV600E/C-RAF inhibitor. Considering the incorporation of a methylene bridge between the terminal phenyl and cyclic diamine, focused quinoline-based arylamides have been tailored, synthesized, and biologically evaluated. Among them, the 5/6-hydroxyquinolines 17b and 18a stood out as the most potent members, with IC50 values of 0.128 µM, 0.114 µM against B-RAFV600E, and 0.0653 µM, 0.0676 µM against C-RAF. Most importantly, 17b elicited remarkable inhibitory potency against the clinically resistant B-RAFV600K mutant with an IC50 value of 0.0616 µM. The putative binding mode of 17b and 18a were studied by molecular docking and molecular dynamics (MD). Moreover, the antiproliferative activity of all target compounds has been examined over a panel of NCI-60 human cancer cell lines. In agreement with cell-free assays, the designed compounds exerted superior anticancer impact over the lead quinoline VII against all cell lines at a 10 µM dose. Notably, both 17b and 18b showed highly potent antiproliferative activity against melanoma cell lines with growth percent under -90% (SK-MEL-29, SK-MEL-5, and UACC-62) at a single dose, while 17b maintained potency with GI50 values of 1.60-1.89 µM against melanoma cell lines. Taken together, 17b, a promising B-RAFV600E/V600K and C-RAF kinase inhibitor, may serve as a valuable candidate in the arsenal of anticancer chemotherapeutics.

Adaptation of Rhizosphere Microbial Communities to Continuous Exposure to Multiple Residual Antibiotics in Vegetable Farms.

The constant application of manure-based fertilizers in vegetable farms leads to antibiotic residue accumulation in soils, which has become a major stressor affecting agroecosystem stability. The present study investigated the adaptation profiles of rhizosphere microbial communities in different vegetable farms to multiple residual antibiotics. Multiple antibiotics, including trimethoprim, sulfonamides, quinolones, tetracyclines, macrolides, lincomycins, and chloramphenicols, were detected in the vegetable farms; the dominant antibiotic (trimethoprim) had a maximum concentration of 36.7 ng/g. Quinolones and tetracyclines were the most prevalent antibiotics in the vegetable farms. The five most abundant phyla in soil samples were Proteobacteria, Actinobacteria, Acidobacteria, Chloroflexi and Firmicutes, while the five most abundant phyla in root samples were Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes and Myxococcota. Macrolides were significantly correlated with microbial community composition changes in soil samples, while sulfonamides were significantly correlated with microbial community composition changes in root samples. Soil properties (total carbon and nitrogen contents and pH) influenced the shifts in microbial communities in rhizosphere soils and roots. This study provides evidence that low residual antibiotic levels in vegetable farms can shift microbial community structures, potentially affecting agroecosystem stability. However, the degree to which the shift occurs could be regulated by environmental factors, such as soil nutrient conditions.

Role of the Water-Metal Ion Bridge in Quinolone Interactions with Escherichia coli Gyrase.

Fluoroquinolones are an important class of antibacterials, and rising levels of resistance threaten their clinical efficacy. Gaining a more full understanding of their mechanism of action against their target enzymes-the bacterial type II topoisomerases gyrase and topoisomerase IV-may allow us to rationally design quinolone-based drugs that overcome resistance. As a step toward this goal, we investigated whether the water-metal ion bridge that has been found to mediate the major point of interaction between Escherichia coli topoisomerase IV and Bacillus anthracis topoisomerase IV and gyrase, as well as Mycobacterium tuberculosis gyrase, exists in E. coli gyrase. This is the first investigation of the water-metal ion bridge and its function in a Gram-negative gyrase. Evidence suggests that the water-metal ion bridge does exist in quinolone interactions with this enzyme and, unlike the Gram-positive B. anthracis gyrase, does use both conserved residues (serine and acidic) as bridge anchors. Furthermore, this interaction appears to play a positioning role. These findings raise the possibility that the water-metal ion bridge is a universal point of interaction between quinolones and type II topoisomerases and that it functions primarily as a binding contact in Gram-positive species and primarily as a positioning interaction in Gram-negative species. Future studies will explore this possibility.

Generation of Aurachin Derivatives by Whole-Cell Biotransformation and Evaluation of Their Antiprotozoal Properties.

The natural product aurachin D is a farnesylated quinolone alkaloid, which is known to possess activity against the causative agent of malaria, Plasmodium spp. In this study, we show that aurachin D inhibits other parasitic protozoa as well. While aurachin D had only a modest effect on Trypanosoma brucei rhodesiense, two other trypanosomatids, T. cruzi and Leishmania donovani, were killed at low micromolar and nanomolar concentrations, respectively, in an in vitro assay. The determined IC50 values of aurachin D were even lower than those of the reference drugs benznidazole and miltefosine. Due to these promising results, we set out to explore the impact of structural modifications on the bioactivity of this natural product. In order to generate aurachin D derivatives with varying substituents at the C-2, C-6 and C-7 position of the quinolone ring system, we resorted to whole-cell biotransformation using a recombinant Escherichia coli strain capable of aurachin-type prenylations. Quinolone precursor molecules featuring methyl, methoxy and halogen groups were fed to this E. coli strain, which converted the substrates into the desired analogs. None of the generated derivatives exhibited improved antiprotozoal properties in comparison to aurachin D. Obviously, the naturally occurring aurachin D features already a privileged structure, especially for the inhibition of the causative agent of visceral leishmaniasis.

Penicillin and Cefotaxime Resistance of Quinolone-Resistant Neisseria meningitidis Clonal Complex 4821, Shanghai, China, 1965-2020.

Clonal complex 4821 (CC4821) Neisseria meningitidis, usually resistant to quinolones but susceptible to penicillin and third-generation cephalosporins, is increasing worldwide. To characterize the penicillin-nonsusceptible (PenNS) meningococci, we analyzed 491 meningococci and 724 commensal Neisseria isolates in Shanghai, China, during 1965-2020. The PenNS proportion increased from 0.3% in 1965-1985 to 7.0% in 2005-2014 and to 33.3% in 2015-2020. Of the 26 PenNS meningococci, 11 (42.3%) belonged to the CC4821 cluster; all possessed mutations in penicillin-binding protein 2, mostly from commensal Neisseria. Genetic analyses and transformation identified potential donors of 6 penA alleles. Three PenNS meningococci were resistant to cefotaxime, 2 within the CC4821 cluster. With 96% of the PenNS meningococci beyond the coverage of scheduled vaccination and the cefotaxime-resistant isolates all from toddlers, quinolone-resistant CC4821 has acquired penicillin and cefotaxime resistance closely related to the internationally disseminated ceftriaxone-resistant gonococcal FC428 clone, posing a greater threat especially to young children.

Identification of 2-Aryl-Quinolone Inhibitors of Cytochrome bd and Chemical Validation of Combination Strategies for Respiratory Inhibitors against Mycobacterium tuberculosis.

Mycobacterium tuberculosis cytochrome bd quinol oxidase (cyt bd), the alternative terminal oxidase of the respiratory chain, has been identified as playing a key role during chronic infection and presents a putative target for the development of novel antitubercular agents. Here, we report confirmation of successful heterologous expression of M. tuberculosis cytochrome bd. The heterologous M. tuberculosis cytochrome bd expression system was used to identify a chemical series of inhibitors based on the 2-aryl-quinolone pharmacophore. Cytochrome bd inhibitors displayed modest efficacy in M. tuberculosis growth suppression assays together with a bacteriostatic phenotype in time-kill curve assays. Significantly, however, inhibitor combinations containing our front-runner cyt bd inhibitor CK-2-63 with either cyt bcc-aa3 inhibitors (e.g., Q203) and/or adenosine triphosphate (ATP) synthase inhibitors (e.g., bedaquiline) displayed enhanced efficacy with respect to the reduction of mycobacterium oxygen consumption, growth suppression, and in vitro sterilization kinetics. In vivo combinations of Q203 and CK-2-63 resulted in a modest lowering of lung burden compared to treatment with Q203 alone. The reduced efficacy in the in vivo experiments compared to in vitro experiments was shown to be a result of high plasma protein binding and a low unbound drug exposure at the target site. While further development is required to improve the tractability of cyt bd inhibitors for clinical evaluation, these data support the approach of using small-molecule inhibitors to target multiple components of the branched respiratory chain of M. tuberculosis as a combination strategy to improve therapeutic and pharmacokinetic/pharmacodynamic (PK/PD) indices related to efficacy.

Identification of qnrVF in a Multidrug-Resistant Vibrio furnissii Clinical Strain.

We found a new qnr gene, qnrVF1, carried by a multidrug resistance plasmid in a clinical Vibrio furnissii isolate. QnrVF1 exhibits 44.6% to 72.5% similarity in identity with other Qnr family proteins. QnrVF alleles are mainly encoded by chromosomes of V. furnissii and Vibrio fluvialis. Phylogenic analysis showed that QnrVF1 and QnrVF2 form a distinct clade in Qnr proteins. Thus, qnrVF represents a new qnr family. In addition, the qnrVF1 gene is often flanked by the mobile element ISCR1. Thus, it is likely that qnrVF1 is mobilized by ISCR1 from chromosome to plasmid in V. furnissii. IMPORTANCE Quinolones are widely used drugs. Bacteria contain a quinolone resistance gene, which mediates resistance to quinolones. Currently, seven families of Qnr proteins, QnrVC, QnrA, QnrB, QnrC, QnrD, QnrE, and QnrS, have been identified. However, it is unclear whether there are any other qnr families. In this study, we identified a new qnr family, qnrVF. We found many V. furnissii and V. fluvialis strains that possess chromosomal qnrVF alleles, suggesting that V. furnissii and V. fluvialis are the reservoirs of qnrVF. We also found that QnrVF1 confers low-level resistance to quinolones. ISCR1 may facilitate the spread of qnrVF1. The emergence and spread of qnrVF may pose a considerable threat to public health.

Molecular characterization of antimicrobial resistance related genes in E. coli, Salmonella and Klebsiella isolates from broilers in the West Region of Cameroon.

BACKGROUND: Antibiotic resistance has become an enduring threat to human health. This has prompted extensive research to identify the determinants responsible in a bid to fight the spread of resistance and also develop new antibiotics. However, routine procedures focus on identifying genetic determinants of resistance only on phenotypically resistant isolates. We aimed to characterise plasmid mediated resistance determinants in key Enterobacteriaceae isolates with differential phenotypic susceptibility profiles and evaluated the contribution of resistance genes on phenotypic expression of susceptibility. METHODS: The study was carried out on 200 Enterobacteriaceae isolates belonging to the genera E. coli, Salmonella, and Klebsiella; 100 resistant and 100 susceptible to quinolones, aminoglycosides, and ESBL-producing as determined by disk diffusion. Reduced susceptibility in susceptible isolates was determined as an increased MIC by broth microdilution. Plasmid-borne resistance genes were sought in all isolates by endpoint PCR. We performed correlations tests to determine the relationship between the occurrence of resistance genes and increased MIC in susceptible isolates. We then used the notion of penetrance to show adequacy between resistance gene carriage and phenotypic resistance as well as diagnostic odds ratio to evaluate how predictable phenotypic susceptibility profile could determine the presence of resistant genes in the isolates. RESULTS: Reduced susceptibility was detected in 30% (9/30) ESBL negative, 50% (20/40) quinolone-susceptible and 53.33% (16/30) aminoglycoside-susceptible isolates. Plasmid-borne resistance genes were detected in 50% (15/30) of ESBL negative, 65% (26/40) quinolone susceptible and 66.67% (20/30) aminoglycoside susceptible isolates. Reduced susceptibility increased the risk of susceptible isolates carrying resistance genes (ORs 4.125, 8.36, and 8.89 respectively for ESBL, quinolone, and aminoglycoside resistance genes). Resistance gene carriage correlated significantly to reduced susceptibility for quinolone and aminoglycoside resistance genes (0.002 and 0.015 at CI95). Gene carriage correlated with phenotypic resistance at an estimated 64.28% for ESBL, 56.90% for quinolone, and 58.33% for aminoglycoside resistance genes. CONCLUSIONS: A high carriage of plasmid-mediated genes for ESBL, quinolone, and aminoglycoside resistance was found among the Enterobacteriaceae tested. However, gene carriage was not always correlated with phenotypic expression. This allows us to suggest that assessing genetic determinants of resistance should not be based on AST profile only. Further studies, including assessing the role of chromosomal determinants will shed light on other factors that undermine antimicrobial susceptibility locally.

A 2.8 Å Structure of Zoliflodacin in a DNA Cleavage Complex with Staphylococcus aureus DNA Gyrase.

Since 2000, some thirteen quinolones and fluoroquinolones have been developed and have come to market. The quinolones, one of the most successful classes of antibacterial drugs, stabilize DNA cleavage complexes with DNA gyrase and topoisomerase IV (topo IV), the two bacterial type IIA topoisomerases. The dual targeting of gyrase and topo IV helps decrease the likelihood of resistance developing. Here, we report on a 2.8 Å X-ray crystal structure, which shows that zoliflodacin, a spiropyrimidinetrione antibiotic, binds in the same DNA cleavage site(s) as quinolones, sterically blocking DNA religation. The structure shows that zoliflodacin interacts with highly conserved residues on GyrB (and does not use the quinolone water-metal ion bridge to GyrA), suggesting it may be more difficult for bacteria to develop target mediated resistance. We show that zoliflodacin has an MIC of 4 µg/mL against Acinetobacter baumannii (A. baumannii), an improvement of four-fold over its progenitor QPT-1. The current phase III clinical trial of zoliflodacin for gonorrhea is due to be read out in 2023. Zoliflodacin, together with the unrelated novel bacterial topoisomerase inhibitor gepotidacin, is likely to become the first entirely novel chemical entities approved against Gram-negative bacteria in the 21st century. Zoliflodacin may also become the progenitor of a new safer class of antibacterial drugs against other problematic Gram-negative bacteria.