Fluoroquinolone resistance ofSerratia marcescens: sucrose, salicylate, temperature, and pH induction of phenotypic resistance

2007 ◽  
Vol 53 (11) ◽  
pp. 1239-1245 ◽  
Author(s):  
Sanela Begic ◽  
Elizabeth A. Worobec
Keyword(s):  
Nalidixic Acid ◽  
Environmental Conditions ◽  
Efflux Pump ◽  
Proton Motive Force ◽  
Motive Force ◽  
Natural Resistance ◽  
Fluoroquinolone Resistance ◽  
Efflux Pump Inhibitor ◽  
Phenotypic Resistance ◽  
Acid Accumulation

Serratia marcescens is a nosocomial agent with a natural resistance to a broad spectrum of antibiotics, making the treatment of its infections very challenging. This study examines the influence of salicylate, sucrose, temperature, and pH variability on membrane permeability and susceptibility of S. marcescens to norfloxacin (hydrophilic fluoroquinolone) and nalidixic acid (hydrophobic quinolone). Resistance of wild-type S. marcescens UOC-67 (ATCC 13880) to norfloxacin and nalidixic acid was assessed by minimal inhibitory concentration (MIC) assays after growth in the presence of various concentrations of sucrose and salicylate and different temperatures and pH values. Norfloxacin and nalidixic acid accumulation was determined in the absence and presence of (i) carbonyl cyanide m-chlorophenylhydrazone (CCCP), a proton-motive-force collapser, and (ii) Phe-Arg β-naphthylamide (PAβN), an efflux pump inhibitor. Accumulation of norfloxacin decreased when S. marcescens was grown in high concentrations of salicylate (8 mmol/L) and sucrose (10% m/v), at high temperature (42 °C), and at pH 6, and it was restored in the presence of CCCP because of the collapse of proton-gradient-dependent efflux in S. marcescens. Although nalidixic acid accumulation was observed, it was not affected by salicylate, sucrose, pH, or temperature changes. In the absence of PAβN, and either in the presence or absence of CCCP, a plateau was reached in the nalidixic acid accumulation for all environmental conditions. With the addition of 20 mg/L PAβN nalidixic acid accumulation is restored for all environmental conditions, suggesting that this quinolone is recognized by a yet to be identified S. marcescens pump that does not use proton motive force as its energy source.

scholarly journals Mechanisms of Fluoroquinolone Resistance in Escherichia coli Isolates from Food-Producing Animals

2011 ◽  
Vol 77 (20) ◽  
pp. 7113-7120 ◽  
Author(s):  
Maria Karczmarczyk ◽  
Marta Martins ◽  
Teresa Quinn ◽  
Nola Leonard ◽  
Séamus Fanning
Keyword(s):  
Escherichia Coli ◽  
Nalidixic Acid ◽  
Target Genes ◽  
Efflux Pump ◽  
Multidrug Resistant ◽  
Fluoroquinolone Resistance ◽  
Efflux Pump Inhibitor ◽  
Content Type ◽  
Field Isolates ◽  
K 12

ABSTRACTEleven multidrug-resistantEscherichia coliisolates (comprising 6 porcine and 5 bovine field isolates) displaying fluoroquinolone (FQ) resistance were selected from a collection obtained from the University Veterinary Hospital (Dublin, Ireland). MICs of nalidixic acid and ciprofloxacin were determined by Etest. All showed MICs of nalidixic acid of >256 μg/ml and MICs of ciprofloxacin ranging from 4 to >32 μg/ml. DNA sequencing was used to identify mutations within the quinolone resistance-determining regions of target genes, and quantitative real-time PCR (qRT-PCR) was used to evaluate the expression of the major porin, OmpF, and component genes of the AcrAB-TolC efflux pump and its associated regulatory loci. Decreased MIC values to nalidixic acid and/or ciprofloxacin were observed in the presence of the efflux pump inhibitor phenylalanine-arginine-β-naphthylamide (PAβN) in some but not all isolates. Several mutations were identified in genes coding for quinolone target enzymes (3 to 5 mutations per strain). All isolates harbored GyrA amino acid substitutions at positions 83 and 87. Novel GyrA (Asp87 → Ala), ParC (Ser80 → Trp), and ParE (Glu460 → Val) substitutions were observed. The efflux activity of these isolates was evaluated using a semiautomated ethidium bromide (EB) uptake assay. Compared to wild-typeE. coliK-12 AG100, isolates accumulated less EB, and in the presence of PAβN the accumulation of EB increased. Upregulation of theacrBgene, encoding the pump component of the AcrAB-TolC efflux pump, was observed in 5 of 11 isolates, while 10 isolates showed decreased expression of OmpF. This study identified multiple mechanisms that likely contribute to resistance to quinolone-based drugs in the field isolates studied.

scholarly journals CCCP experimental evolution ofEscherichia coliselects for mutations that increase EmrA activity or that downregulate other PMF-driven drug efflux pumps

2018 ◽  
Author(s):  
Jessie M. Griffith ◽  
Preston J. Basting ◽  
Katarina M. Bischof ◽  
Erintrude P. Wrona ◽  
Karina S. Kunka ◽  
...  
Keyword(s):  
Experimental Evolution ◽  
Efflux Pump ◽  
Enteric Bacteria ◽  
Efflux Pumps ◽  
Proton Motive Force ◽  
Motive Force ◽  
Drug Efflux ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
K 12

ABSTRACTExperimental evolution was conducted withEscherichia coliK-12 W3110 in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP), an uncoupler of the proton motive force (PMF). Cultures were serially diluted daily 1:100 in broth medium containing 20-150 μM CCCP at pH 6.5 or at pH 8.0. After 1,000 generations, all populations showed 5- to 10-fold increase in CCCP resistance. Sequenced isolates showed mutations inemrABor in its negative repressormprA; the EmrAB-TolC multidrug efflux pump confers resistance to CCCP and nalidixic acid. Deletion ofemrAabolished the CCCP resistance of these strains. One CCCP-evolved isolate lackedemrAormprAmutations; this strain (C-B11-1) showed mutations in drug efflux regulatorscecR(ybiH) (upregulates drug pumps YbhG and YbhFSR) andgadE(upregulates drug pumpmdtEF). AcecR∷kanRdeletion conferred partial resistance to CCCP. A later evolved descendant of the C-B11 population showed mutations inybhR(MDR efflux). Another isolate showedacrB(MDR efflux pump). TheacrBisolate was sensitive to chloramphenicol and tetracycline, which are effluxed by AcrAB. Other mutant genes in CCCP-evolved strains includeadhE(alcohol dehydrogenase),rng(ribonuclease G), andcyaA(adenylate cyclase). Overall, experimental evolution revealed a CCCP fitness advantage for mutations increasing its own efflux via EmrA; and for mutations that may decrease proton-driven pumps that efflux other drugs not present (cecR, gadE, acrB, ybhR). These results are consistent with our previous report of drug sensitivity associated with evolved tolerance to a partial uncoupler (benzoate or salicylate).IMPORTANCEThe genetic responses of bacteria to depletion of proton motive force, and their effects on drug resistance, are poorly understood. Our evolution experiment reveals genetic mechanisms of adaptation to the PMF uncoupler CCCP, including selection for and against various multidrug efflux pumps. The results have implications for our understanding of the gut microbiome, which experiences high levels of organic acids that decrease PMF. Organic acid uncouplers may select against multidrug resistance in evolving populations of enteric bacteria.

scholarly journals Use of an Efflux Pump Inhibitor To Determine the Prevalence of Efflux Pump-Mediated Fluoroquinolone Resistance and Multidrug Resistance in Pseudomonas aeruginosa

2005 ◽  
Vol 49 (2) ◽  
pp. 565-570 ◽  
Author(s):  
Jane Kriengkauykiat ◽  
Edith Porter ◽  
Olga Lomovskaya ◽  
Annie Wong-Beringer
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Multidrug Resistance ◽  
Substrate Specificity ◽  
Efflux Pump ◽  
Fluoroquinolone Resistance ◽  
Cross Resistance ◽  
Broth Microdilution ◽  
Efflux Pump Inhibitor ◽  
Broad Substrate Specificity ◽  
Resistant Strains

ABSTRACT Fluoroquinolone-resistance in Pseudomonas aeruginosa may be due to efflux pump overexpression (EPO) and/or target mutations. EPO can result in multidrug resistance (MDR) due to broad substrate specificity of the pumps. MC-04,124, an efflux pump inhibitor (EPI) shown to significantly potentiate activity of levofloxacin in P. aeruginosa, was used to examine the prevalence of EPO in clinical isolates. MICs were determined for ciprofloxacin, levofloxacin, moxifloxacin, and gatifloxacin with or without EPI and for other antipseudomonal agents by using broth microdilution against P. aeruginosa isolates from adults (n = 119) and children (n = 24). The prevalence of the EPO phenotype (≥8-fold MIC decrease when tested with EPI) was compared among subgroups with different resistance profiles. The EPO phenotype was more prevalent among levofloxacin-resistant than levofloxacin-sensitive strains (61%, 48/79 versus 9%, 6/64). EPO was present in 60% of fluoroquinolone-resistant strains without cross-resistance, while it was present at variable frequencies among strains with cross-resistance to other agents: piperacillin-tazobactam (86%), ceftazidime (76%), cefepime (65%), imipenem (56%), gentamicin (55%), tobramycin (48%), and amikacin (27%). The magnitude of MIC decrease with an EPI paralleled the frequency of which the EPO phenotype was observed in different subgroups. EPI reduced the levofloxacin MIC by as much as 16-fold in eight strains for which MICs were 128 μg/ml. Efflux-mediated resistance appears to contribute significantly to fluoroquinolone resistance and MDR in P. aeruginosa. Our data support the fact that increased fluoroquinolone usage can negatively impact susceptibility of P. aeruginosa to multiple classes of antipseudomonal agents.

scholarly journals The FlgN chaperone activates the Na+-driven engine of the Salmonella flagellar protein export apparatus

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Tohru Minamino ◽  
Miki Kinoshita ◽  
Yusuke V. Morimoto ◽  
Keiichi Namba
Keyword(s):  
Conformational Change ◽  
Environmental Conditions ◽  
Proton Motive Force ◽  
Protein Export ◽  
Motive Force ◽  
Structural Proteins ◽  
Wild Type ◽  
Flagellar Protein

AbstractThe bacterial flagellar protein export machinery consists of a transmembrane export gate complex and a cytoplasmic ATPase complex. The gate complex has two intrinsic and distinct H+-driven and Na+-driven engines to drive the export of flagellar structural proteins. Salmonella wild-type cells preferentially use the H+-driven engine under a variety of environmental conditions. To address how the Na+-driven engine is activated, we analyzed the fliJ(Δ13–24) fliH(Δ96–97) mutant and found that the interaction of the FlgN chaperone with FlhA activates the Na+-driven engine when the ATPase complex becomes non-functional. A similar activation can be observed with either of two single-residue substitutions in FlhA. Thus, it is likely that the FlgN-FlhA interaction generates a conformational change in FlhA that allows it to function as a Na+ channel. We propose that this type of activation would be useful for flagellar construction under conditions in which the proton motive force is severely restricted.

Molecular Characterization of Fluoroquinolone Resistance Mechanisms of Campylobacter Isolates from Duck Meats

2017 ◽  
Vol 80 (12) ◽  
pp. 2056-2059
Author(s):  
Min Kang ◽  
Bai Wei ◽  
Sung-Woon Choi ◽  
Se-Yeoun Cha ◽  
Hyung-Kwan Jang
Keyword(s):  
Nalidixic Acid ◽  
Efflux Pump ◽  
Resistance Mechanisms ◽  
Quinolone Resistance ◽  
Fluoroquinolone Resistance ◽  
Gyra Gene ◽  
Meat Samples ◽  
High Level ◽  
Level Resistance

ABSTRACT The purpose of this study was to identify the molecular basis of quinolone resistance of Campylobacter isolates recovered from duck meats. Sixty-one isolates from duck meat samples were studied using sequence analysis of the gyrA gene, and PCR assays were used to identify the presence of the CmeABC efflux pump and its restored sensitivity in the presence of efflux-pump inhibitors. High-level resistance to nalidixic acid and ciprofloxacin was attributed to amino acid substitutions Thr-86-Ile in some isolates. The PCR assay confirmed the presence of the cmeB gene in 29 (47.5%) of the 61 Campylobacter isolates. Phenylalanine arginine β-naphthylamide reduced the MICs of ciprofloxacin and nalidixic acid in 16 (55.2%) and 26 (89.7%) isolates, respectively. The Thr-86-Ile substitution in the gyrA was the primary contributor to the high-level quinolone resistance in Campylobacter isolates from duck meats.

scholarly journals Acetobacter aceti Possesses a Proton Motive Force-Dependent Efflux System for Acetic Acid

2005 ◽  
Vol 187 (13) ◽  
pp. 4346-4352 ◽  
Author(s):  
Kazunobu Matsushita ◽  
Taketo Inoue ◽  
Osao Adachi ◽  
Hirohide Toyama
Keyword(s):  
Acetic Acid ◽  
Efflux Pump ◽  
Membrane Vesicles ◽  
Proton Motive Force ◽  
Motive Force ◽  
Efflux System ◽  
Acetobacter Aceti ◽  
Intact Cells ◽  
Inside Out ◽  
Acetic Acid Acetate

ABSTRACT Acetic acid bacteria are obligate aerobes able to oxidize ethanol, sugar alcohols, and sugars into their corresponding acids. Among them, Acetobacter and Gluconacetobacter species have very high ethanol oxidation capacity, leading to accumulation of vast amounts of acetic acid outside the cell. Since these bacteria are able to grow in media with high concentrations of acetic acid, they must possess a specific mechanism such as an efflux pump by which they can resist the toxic effects of acetic acid. In this study, the efflux pump of Acetobacter aceti IFO 3283 was examined using intact cells and membrane vesicles. The accumulation of acetic acid/acetate in intact cells was increased by the addition of a proton uncoupler and/or cyanide, suggesting the presence of an energy-dependent efflux system. To confirm this, right-side-out and inside-out membrane vesicles were prepared from A. aceti IFO 3283, and the accumulation of acetic acid/acetate in the vesicles was examined. Upon the addition of a respiratory substrate, the accumulation of acetic acid/acetate in the right-side-out vesicles was largely decreased, while its accumulation was very much increased in the inside-out vesicles. These respiration-dependent phenomena observed in both types of membrane vesicles were all sensitive to a proton uncoupler. Acetic acid/acetate uptake in the inside-out membrane vesicles was dependent not on ATP but on the proton motive force. Furthermore, uptake was shown to be rather specific for acetic acid and to be pH dependent, because higher uptake was observed at lower pH. Thus, A. aceti IFO 3283 possesses a proton motive force-dependent efflux pump for acetic acid.

scholarly journals A small RNA controls bacterial resistance to gentamicin during iron starvation

2018 ◽  
Author(s):  
Sylvia Chareyre ◽  
Frédéric Barras ◽  
Pierre Mandin
Keyword(s):  
Escherichia Coli ◽  
Iron Homeostasis ◽  
Bacterial Resistance ◽  
Proton Motive Force ◽  
Motive Force ◽  
Regulatory Rna ◽  
Iron Starvation ◽  
Phenotypic Resistance ◽  
Respiratory Complexes ◽  
Small Regulatory Rna

ABSTRACTPhenotypic resistance describes a bacterial population that becomes transiently resistant to an antibiotic without requiring a genetic change. We here investigated the role of the small regulatory RNA (sRNA) RyhB, a key contributor to iron homeostasis, in the phenotypic resistance ofEscherichia colito various classes of antibiotics. We found that RyhB induces resistance to gentamicin, an aminoglycoside that targets the ribosome, when iron is scarce. RyhB induced resistance is due to the inhibition of respiratory complexes Nuo and Sdh activities. These complexes, which contain numerous Fe-S clusters, are crucial for generating a proton motive force (pmf) that allows gentamicin uptake. RyhB directly represses the expression ofnuoandsdhoperons by binding to their mRNAs, thereby inhibiting their translation. Indirectly, RyhB also inhibits the maturation of Nuo and Sdh by repressing synthesis of the Isc Fe-S biogenesis machinery. Notably, our study identifiesnuoas a new direct RyhB target and shows that respiratory complexes activity levels are predictive of the bacterial sensitivity to gentamicin. Altogether, these results unveil a new role for RyhB in the adaptation to antibiotic stress, an unprecedented consequences of its role in iron starvation stress response.AUTHOR’S SUMMARYUnderstanding the mechanisms at work behind bacterial antibiotic resistance has become a major health issue in the face of the antibiotics crisis. Here, we show that RyhB, a bacterial small regulatory RNA, induces resistance ofEscherichia colito the antibiotic gentamicin when iron is scarce, an environmental situation prevalent during host-pathogen interactions. This resistance is due to RyhB repression of the synthesis and post-translational maturation of the respiratory complexes Nuo and Sdh. These complexes are crucial in producing the proton motive force that allows uptake of the antibiotics in the cell. Altogether, these data point out to a major role for RyhB in escaping antibacterial action.

Quinolone-resistant Salmonella Typhi in South Africa, 2003–2007

2009 ◽  
Vol 138 (1) ◽  
pp. 86-90 ◽  
Author(s):  
A. M. SMITH ◽  
N. GOVENDER ◽  
K. H. KEDDY
Keyword(s):  
South Africa ◽  
Amino Acid ◽  
Nalidixic Acid ◽  
Efflux Pump ◽  
Variable Number Tandem Repeat ◽  
Quinolone Resistance ◽  
Efflux Pump Inhibitor ◽  
Pcr Screening ◽  
Active Efflux ◽  
Amino Acid Mutations

SUMMARYIn South Africa, for the years 2003–2007, the Enteric Diseases Reference Unit received 510 human isolates of Salmonella Typhi, of which 27 were nalidixic acid-resistant [minimum inhibitory concentrations (MICs) 128–512 μg/ml] with reduced susceptibility to ciprofloxacin (MICs 0·125–0·5 μg/ml). Pulsed-field gel electrophoresis analysis of 19 available isolates differentiated them into five DNA pattern types; multiple-locus variable-number tandem repeat analysis differentiated the isolates into 10 types. This level of genetic diversity suggested that resistant strains usually emerged independently of one another. A 16- to 32-fold decrease in nalidixic acid MIC and a 2- to 8-fold decrease in ciprofloxacin MIC, was observed in the presence of an efflux pump inhibitor. All isolates were negative by PCR screening for qnr genes. Seven resistant isolates were further analysed for mutations in the quinolone resistance-determining region of gyrA, gyrB, parC and parE. No amino-acid mutations were identified in GyrB and ParE; all isolates showed amino-acid mutations in both GyrA and ParC. We conclude that amino-acid mutations in GyrA and ParC in combination with active efflux of antibiotic out of the bacterial cell are the probable mechanisms conferring quinolone resistance.

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