scholarly journals Mutations that increase expression of the EmrAB-TolC efflux pump confer increased resistance to nitroxoline in Escherichia coli

2019 ◽  
Author(s):  
Fabiola Puértolas-Balint ◽  
Omar Warsi ◽  
Marius Linkevicius ◽  
Po-Cheng Tang ◽  
Dan I Andersson
Keyword(s):  
Escherichia Coli ◽  
Target Genes ◽  
Efflux Pump ◽  
Resistance Mechanisms ◽  
Cross Resistance ◽  
Resistance Mutations ◽  
Double Knockout ◽  
Low Level ◽  
Resistant Mutants ◽  
Level Resistance

Abstract Objectives To determine the mechanism of resistance to the antibiotic nitroxoline in Escherichia coli. Methods Spontaneous nitroxoline-resistant mutants were selected at different concentrations of nitroxoline. WGS and strain reconstruction were used to define the genetic basis for the resistance. The mechanistic basis of resistance was determined by quantitative PCR (qPCR) and by overexpression of target genes. Fitness costs of the resistance mutations and cross-resistance to other antibiotics were also determined. Results Mutations in the transcriptional repressor emrR conferred low-level resistance to nitroxoline [nitroxoline MIC (MICNOX) = 16 mg/L] by increasing the expression of the emrA and emrB genes of the EmrAB-TolC efflux pump. These resistant mutants showed no fitness reduction and displayed cross-resistance to nalidixic acid. Second-step mutants with higher-level resistance (MICNOX = 32–64 mg/L) had mutations in the emrR gene, together with either a 50 kb amplification, a mutation in the gene marA, or an IS upstream of the lon gene. The latter mutations resulted in higher-level nitroxoline resistance due to increased expression of the tolC gene, which was confirmed by overexpressing tolC from an inducible plasmid in a low-level resistance mutant. Furthermore, the emrR mutations conferred a small increase in resistance to nitrofurantoin only when combined with an nfsAB double-knockout mutation. However, nitrofurantoin-resistant nfsAB mutants showed no cross-resistance to nitroxoline. Conclusions Mutations in different genes causing increased expression of the EmrAB-TolC pump lead to an increased resistance to nitroxoline. The structurally similar antibiotics nitroxoline and nitrofurantoin appear to have different modes of action and resistance mechanisms.

Pyrimethanil Sensitivity and Resistance Mechanisms in Penicillium digitatum

Plant Disease ◽  
2020 ◽  
Author(s):  
Yuchao Zhang ◽  
Yanping Fu ◽  
Chaoxi Luo ◽  
Fuxing Zhu
Keyword(s):  
Target Genes ◽  
Sodium Dodecyl ◽  
Resistance Mechanisms ◽  
Penicillium Digitatum ◽  
Cross Resistance ◽  
Postharvest Diseases ◽  
Cell Wall Degrading Enzymes ◽  
Baseline Sensitivity ◽  
Nucleotide Mutation ◽  
Resistant Mutants

Pyrimethanil is an anilinopyrimidines (AP) fungicide and highly effective in controlling green mold caused by Penicillium digitatum but has not yet been registered in China to control postharvest diseases of citrus. In the present study, baseline sensitivity of P. digitatum to pyrimethanil was established based on the effective concentrations for 50% inhibition (EC50) values of 127 isolates collected from five major citrus-growing regions of China. The distribution of these EC50 values was unimodal but with a long right tail. The mean EC50 value was 0.137 ± 0.046 μg/mL (SD), and the minimum and maximum were 0.073 and 0.436 μg/mL, respectively. Pyrimethanil in potato dextrose agar (PDA) at 0.20 μg/mL decreased methionine production in the mycelia by 21.6% and reduced the activities of cell wall-degrading enzymes cellulase and pectinase by 9.1 and 32.8%, respectively. Twelve pyrimethanil-resistant mutants were obtained by consecutive sub-culturing of 12 arbitrarily selected sensitive isolates on pyrimethanil-amended PDA for 4 generations, and the resistance factors ranged from 69 to 3421. There was no cross-resistance between pyrimethanil and prochloraz (r = 0.377, P = 0.123). Compared with their parental isolates, pyrimethanil-resistant mutants had reduced pathogenicity to citrus fruit but higher tolerance to hydrogen peroxide. No differences were detected in tolerance to NaCl, CaCl2, Congo red, or sodium dodecyl sulfate (SDS). Exogenous addition of methionine into PDA partially alleviated the toxicity of pyrimethanil to the sensitive isolates but had no significant effect on toxicity to the resistant mutants. Sequencing of cystathionine γ-synthase encoding genes CGS1 and CGS2, the potential target genes for pyrimethanil, showed that there was no nucleotide mutation in the coding region of CGS of the pyrimethanil-resistant mutants. However, the relative expression of CGS1 and CGS2 genes of the pyrimethanil-resistant mutants was reduced by 42.5 and 57.4%, respectively. These results have important implications for applications of pyrimethanil to control P. digitatum and for understanding the modes of action and resistance mechanisms of pyrimethanil.

scholarly journals The Role of Efflux Pumps in the Transition from Low-Level to Clinical Antibiotic Resistance

Antibiotics ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 855
Author(s):  
Anna Elisabeth Ebbensgaard ◽  
Anders Løbner-Olesen ◽  
Jakob Frimodt-Møller
Keyword(s):  
Antibiotic Resistance ◽  
Target Genes ◽  
Defense Mechanism ◽  
Efflux Pump ◽  
Spontaneous Mutation ◽  
Efflux Pumps ◽  
Infection Site ◽  
Low Level ◽  
Clinical Resistance ◽  
Level Resistance

Antibiotic resistance is on the rise and has become one of the biggest public health challenges of our time. Bacteria are able to adapt to the selective pressure exerted by antibiotics in numerous ways, including the (over)expression of efflux pumps, which represents an ancient bacterial defense mechanism. Several studies show that overexpression of efflux pumps rarely provides clinical resistance but contributes to a low-level resistance, which allows the bacteria to persist at the infection site. Furthermore, recent studies show that efflux pumps, apart from pumping out toxic substances, are also linked to persister formation and increased spontaneous mutation rates, both of which could aid persistence at the infection site. Surviving at the infection site provides the low-level-resistant population an opportunity to evolve by acquiring secondary mutations in antibiotic target genes, resulting in clinical resistance to the treating antibiotic. Thus, this emphasizes the importance and challenge for clinicians to be able to monitor overexpression of efflux pumps before low-level resistance develops to clinical resistance. One possible treatment option could be an efflux pump-targeted approach using efflux pump inhibitors.

scholarly journals Clinical resistance to erythromycin and clindamycin in cutaneous propionibacteria isolated from acne patients is associated with mutations in 23S rRNA.

1997 ◽  
Vol 41 (5) ◽  
pp. 1162-1165 ◽  
Author(s):  
J I Ross ◽  
E A Eady ◽  
J H Cove ◽  
C E Jones ◽  
A H Ratyal ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genetic Basis ◽  
23S Rrna ◽  
Cross Resistance ◽  
Peptidyl Transferase ◽  
Erythromycin Resistance ◽  
Clinical Resistance ◽  
Resistant Mutants ◽  
High Level ◽  
Level Resistance

The genetic basis of erythromycin resistance in cutaneous propionibacteria was determined by comparing the nucleotide sequences of the peptidyl transferase region in the 23S rRNAs from 9 susceptible and 26 resistant clinical isolates as well as 4 laboratory-selected erythromycin-resistant mutants of a susceptible strain. In 13 isolates and the 4 laboratory mutants, cross-resistance to macrolides, lincosamides, and B-type streptogramins was associated with an A-->G transition at a position cognate with Escherichia coli 23S rRNA base 2058. These strains were resistant to > or = 512 microg of erythromycin per ml. Two other mutations were identified, an A-->G transition at base 2059 in seven strains, associated with high-level resistance to all macrolides, and a G-->A transition at base 2057 in six strains, associated with low-level resistance to erythromycin. These mutations correspond to three of four phenotypic classes previously identified by using MIC determinations.

scholarly journals Characterization of Triclosan-Resistant Mutants Reveals Multiple Antimicrobial Resistance Mechanisms in Rhodospirillum rubrum S1H

2010 ◽  
Vol 76 (10) ◽  
pp. 3116-3123 ◽  
Author(s):  
Benny F. G. Pycke ◽  
Aurélie Crabbé ◽  
Willy Verstraete ◽  
Natalie Leys
Keyword(s):  
Antimicrobial Resistance ◽  
Point Mutation ◽  
Defense Mechanisms ◽  
Rhodospirillum Rubrum ◽  
Efflux Pump ◽  
Resistance Mechanisms ◽  
Cross Resistance ◽  
Cell Permeabilization ◽  
Initial Exposure ◽  
Low Level

ABSTRACT Antimicrobial resistance mechanisms were identified in 11 spontaneous high- and low-level triclosan resistance (Tcsr) mutants of Rhodospirillum rubrum S1H by genotyping complemented with transcriptional analyses, antibiotic resistance screening, and membrane permeability analyses. High-end Tcsr (MIC = 8 mg/liter) was the result of a FabI1(G98V) mutation. This point mutation led to an even higher level of Tcsr (MIC ≥ 16 mg/liter) in combination with constitutive upregulation of mexB and mexF efflux pump homologs. Hence, a mechanistic synergy of constitutive efflux pump expression and a FabI1 point mutation could prevent TCS-induced cell permeabilization, which was shown to occur between 4 and 8 mg/liter TCS in the R. rubrum S1H parent strain. Low-level Tcsr mutants constitutively upregulated the emrAB, mexAB, and/or mexF homolog. The mutants that overexpressed emrAB also derepressed the micropollutant-upregulated factors mufA1 and mufM. In some cases, low-level Tcsr decreased innate resistance to ampicillin and tetracycline, while in others, a triclosan-induced antibiotic cross-resistance was shown for chloramphenicol and carbenicillin. This study showed that the TCS resistance degree is dependent of the initial exposure concentration in Rhodospirillum rubrum S1H and that similar resistance degrees can be the result of different defense mechanisms, which all have distinct antibiotic cross-resistance profiles.

scholarly journals Efflux mediated chlorpyrifos tolerance in Escherichia coli BL21(DE3)

2020 ◽  
Author(s):  
Aswathi Aswathi ◽  
Ashok Pandey ◽  
Rajeev K Sukumaran
Keyword(s):  
Escherichia Coli ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Resistance Mechanisms ◽  
Cross Resistance ◽  
Antibiotics Resistance ◽  
High Tolerance ◽  
Intrinsic Resistance ◽  
Efflux Pump Inhibitor ◽  
Environmental Strain

AbstractBacteria are continually challenged with variety of synthetic chemicals/xenobiotics in their immediate surroundings, including pesticides. Chlorpyrifos is one of the most commonly used organophosphate pesticides in the world. The non-environmental strain of Escherichia coli, BL21 (DE3) displayed high tolerance to chlorpyrifos but with no/negligible degradation. The intrinsic resistance mechanisms that aid the organism in its high tolerance are probed. Efflux pumps being ubiquitous in nature and capable of conferring resistance against wide variety of xenobiotics were found to be over-expressed in the presence of CP. Also, an efflux pump inhibitor PAβN increased the susceptibility of E. coli to chlorpyrifos due to the intracellular accumulation of CP. The tripartite efflux pump EmrAB-TolC with increased expression in both transcript and protein on CP exposure, might play a major role in CP tolerance. The transcriptional regulators involved in multidrug resistance along with transporters belonging to all the major families conferring antimicrobial resistance were up-regulated. Also up-regulated were the genes involved in phopshonate metabolism and all the genes in the copper or silver export system. The common resistance mechanisms i.e, activation of efflux pumps between CP, antibacterial metals and antibiotics resistance might result in cross-resistance, ultimately increasing the prevalence of multidrug resistant strains, making infections hard to treat.

scholarly journals Role of low-level quinolone resistance in generating tolerance in Escherichia coli under therapeutic concentrations of ciprofloxacin

2020 ◽  
Author(s):  
M Ortiz-Padilla ◽  
S Diaz-Diaz ◽  
J Machuca ◽  
A Tejada-Gonzalez ◽  
E Recacha ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Sos Response ◽  
Resistance Mechanisms ◽  
Quinolone Resistance ◽  
Response Suppression ◽  
Bacterial Survival ◽  
Resistance Mutations ◽  
Low Level ◽  
Short Period ◽  
The Impact

Abstract Background Tolerance (including persistence) and resistance result in increased survival under antibiotic pressure. Objectives We evaluated the interplay between resistance and tolerance to ciprofloxacin under therapeutic and killing conditions to determine the contribution of low-level quinolone resistance (LLQR) mechanisms to tolerance. We also determined how the interaction between resistance (LLQR phenotypes) and tolerance was modified under SOS response suppression. Methods Twelve isogenic Escherichia coli strains harbouring quinolone resistance mechanisms combined with SOS response deficiency and six clinical E. coli isolates (LLQR or non-LLQR) were evaluated. Survival (tolerance or persistence) assays were used to measure surviving bacteria after a short period (up to 4 h) of bactericidal antibiotic treatment under therapeutic and killing concentrations of ciprofloxacin [1 mg/L, EUCAST/CLSI breakpoint for resistance; and 2.5 mg/L, peak serum concentration (Cmax) of this drug]. Results QRDR substitutions (S83L in GyrA alone or combined with S80R in ParC) significantly increased the fraction of tolerant bacteria (2–4 log10 cfu/mL) after exposure to ciprofloxacin at clinically relevant concentrations. The impact on tolerant bacteria due to SOS response suppression (including persistence mediated by the tisB gene) was reversed by LLQR mechanisms at therapeutic concentrations. Furthermore, no reduction in the fraction of tolerant bacteria due to SOS response suppression was observed when S83L in GyrA plus S80R in ParC were combined. Conclusions Tolerance and quinolone resistance mutations interact synergistically, giving LLQR mechanisms an additional role in allowing bacterial survival and evasion of therapeutic antimicrobial conditions by a combination of the two strategies. At clinically relevant concentrations, LLQR mechanisms reverse further impact of SOS response suppression in reducing bacterial tolerance.

scholarly journals Mutations That Enhance the Ciprofloxacin Resistance of Escherichia coli withqnrA1

2015 ◽  
Vol 60 (3) ◽  
pp. 1537-1545 ◽  
Author(s):  
Laura Vinué ◽  
Marian A. Corcoran ◽  
David C. Hooper ◽  
George A. Jacoby
Keyword(s):  
Escherichia Coli ◽  
Inner Core ◽  
Efflux Pump ◽  
Dna Gyrase ◽  
Resistance Mutations ◽  
Content Type ◽  
E Coli ◽  
Ciprofloxacin Resistance ◽  
Level Resistance ◽  
Selection Of

Plasmid-mediatedqnrgenes provide only a modest decrease in quinolone susceptibility but facilitate the selection of higher-level resistance. InEscherichia colistrain J53 withoutqnr, ciprofloxacin resistance often involves mutations in the GyrA subunit of DNA gyrase. Mutations ingyrAwere absent, however, when 43 mutants with decreased ciprofloxacin susceptibility were selected from J53(pMG252) withqnrA1. Instead, in 13 mutants, individual and whole-genome sequencing identified mutations inmarRandsoxRassociated with increased expression ofmarAandsoxSand, through them, increased expression of the AcrAB pump, which effluxes quinolones. Nine mutants had increased expression of the MdtE efflux pump, and six demonstrated increased expression of theydhEpump gene. Many efflux mutants also had increased resistance to novobiocin, another pump substrate, but other mutants were novobiocin hypersusceptible. Mutations inrfaDandrfaEin the pathway for inner core lipopolysaccharide (LPS) biosynthesis were identified in five such strains. Many of the pump and LPS mutants had decreased expression of OmpF, the major porin channel for ciprofloxacin entry. Three mutants had increased expression ofqnrAthat persisted when pMG252 from these strains was outcrossed.gyrAmutations were also rare when mutants with decreased ciprofloxacin susceptibility were selected fromE. coliJ53 withaac(6′)-Ib-crorqepA. We suggest that multiple genes conferring low-level resistance contribute to enhanced ciprofloxacin resistance selected from anE. colistrain carryingqnrA1,aac(6′)-Ib-cr, orqepAbecause these determinants decrease the effective ciprofloxacin concentration and allow more common but lower-resistance mutations than those ingyrAto predominate.

scholarly journals Resistance against Membrane-Inserting MmpL3 Inhibitor through Upregulation of MmpL5 in Mycobacterium tuberculosis

2020 ◽  
Vol 64 (12) ◽  
Author(s):  
Ming Li ◽  
Samuel Agyei Nyantakyi ◽  
Mei-Lin Go ◽  
Thomas Dick
Keyword(s):  
Antibacterial Activity ◽  
Mycobacterium Tuberculosis ◽  
Mouse Model ◽  
Mannich Base ◽  
Efflux Pump ◽  
Transcriptional Repressor ◽  
Mannich Bases ◽  
Mycolic Acid ◽  
Low Level ◽  
Level Resistance

ABSTRACT Spiroketal indolyl Mannich bases (SIMBs) present a novel class of membrane-inserting antimycobacterials with efficacy in a tuberculosis mouse model. SIMBs exert their antibacterial activity by two mechanisms. The indolyl Mannich base scaffold causes permeabilization of bacteria, and the spiroketal moiety contributes to inhibition of the mycolic acid transporter MmpL3. Here, we show that low-level resistance to SIMBs arises by mutations in the transcriptional repressor MmpR5, resulting in upregulation of the efflux pump MmpL5.

scholarly journals Differing Effects of Combination Chemotherapy with Meropenem and Tobramycin on Cell Kill and Suppression of Resistance of Wild-Type Pseudomonas aeruginosa PAO1 and Its Isogenic MexAB Efflux Pump-Overexpressed Mutant

2009 ◽  
Vol 53 (6) ◽  
pp. 2266-2273 ◽  
Author(s):  
G. L. Drusano ◽  
Weiguo Liu ◽  
Christine Fregeau ◽  
Robert Kulawy ◽  
Arnold Louie
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Drug Interaction ◽  
Combination Chemotherapy ◽  
Drug Exposure ◽  
Inhibitory Concentration ◽  
Efflux Pump ◽  
Area Under The Curve ◽  
Resistance Mechanisms ◽  
Wild Type ◽  
Resistant Mutants

ABSTRACT The drug interaction terminology (synergy, additivity, antagonism) relates to bacterial kill. The suppression of resistance requires greater drug exposure. We examined the combination of meropenem and tobramycin for kill and resistance suppression (wild-type Pseudomonas aeruginosa PAO1 and its isogenic MexAB-overexpressed mutant). The drug interaction was additive. The introduction of MexAB overexpression significantly altered the 50% inhibitory concentration of meropenem but not that of tobramycin, resulting in the recovery of a marked increase in colony numbers from drug-containing plates. For the wild type, more tobramycin-resistant isolates than meropenem-resistant isolates were present, and the tobramycin-resistant isolates were harder to suppress. MexAB overexpression unexpectedly caused a significant increase in the number of tobramycin-resistant mutants, as indexed to the area under the curve of slices through the inverted U resistance mountain. The differences were significant, except in the absence of meropenem. We hypothesize that the pump resulted in the presence of less meropenem for organism inhibition, allowing more rounds of replication and also affecting the numbers of tobramycin-resistant mutants. When resistance suppression is explored by combination chemotherapy, it is important to examine the impacts of differing resistance mechanisms for both agents.

Quinoline resistance mechanisms in Plasmodium falciparum: the debate goes on

Parasitology ◽  
1997 ◽  
Vol 114 (7) ◽  
pp. 125-136 ◽  
Author(s):  
S. A. WARD ◽  
P. G. BRAY ◽  
S. R. HAWLEY
Keyword(s):  
Efflux Pump ◽  
Resistance Mechanism ◽  
Resistance Mechanisms ◽  
Food Vacuole ◽  
Drug Binding ◽  
Drug Uptake ◽  
Cross Resistance ◽  
Parasite Resistance ◽  
Therapeutic Success ◽  
Drug Binding Site

Despite considerable therapeutic success with the antimalarial 4-aminoquinolines such as chloroquine, there is serious doubt about the future of this drug class due mainly to the development and spread of parasite resistance throughout endemic areas. In this article we review the possible biochemical and molecular basis of resistance. Based on our current understanding we have considered the possibility of developing strategies which may allow the aminoquinolines to once again be used effectively against P. falciparum. Our conclusions are that drug resistance is the result of a reduced rate of drug uptake which in turn reduces the amount of drug available to bind the target. The basis for this reduced accumulation could be an altered pH gradient making the food vacuole more alkaline or the parasite cytosol more acidic, an efflux pump removing drug directly from the membrane or any other process which will reduce the rate of drug uptake. Central to the effectiveness of this resistance mechanism is the transient availability of a high affinity, low capacity drug binding site (possibly haem) within the parasite. Resistance reversers such as verapamil influence the apparent Ka for this drug binding phenomenon via an increased drug uptake rate. We demonstrate that by chemical modification of the aminoquinolines, producing predictable alterations in their physicochemical properties, that it is possible to minimise the verapamil sensitive component of resistance and reduce significantly cross-resistance patterns without loss in absolute activity. Based on these views we suggest that the aminoquinoline antimalarials still have a role to play in the cheap, safe and effective chemotherapy of falciparum malaria.

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