Tigecycline Efflux as a Mechanism for Nonsusceptibility in Acinetobacter baumannii

ABSTRACT Tigecycline has an extended spectrum of in vitro antimicrobial activities, including that against multidrug-resistant Acinetobacter. After identifying bloodstream isolates of Acinetobacter with reduced susceptibilities to tigecycline, we performed a study to assess tigecycline efflux mediated by the resistance-nodulation-division-type transporter AdeABC. After exposure of two tigecycline-nonsusceptible isolates to the efflux pump inhibitor phenyl-arginine-β-naphthylamide (PABN), a fourfold reduction in the tigecycline MIC was observed. Both tigecycline-susceptible and -nonsusceptible isolates were found to carry the gene coding for the transmembrane component of the AdeABC pump, adeB, and the two-component regulatory system comprising adeS and adeR. Previously unreported point mutations were identified in the regulatory system in tigecycline-nonsusceptible isolates. Real-time PCR identified 40-fold and 54-fold increases in adeB expression in the two tigecycline-nonsusceptible isolates compared to that in a tigecycline-susceptible isolate. In vitro exposure of a tigecycline-susceptible clinical strain to tigecycline caused a rapid rise in the MIC of tigecycline from 2 μg/ml to 24 μg/ml, which was reversible with PABN. A 25-fold increase in adeB expression was observed in a comparison between this tigecycline-susceptible isolate and its isogenic tigecycline-nonsusceptible mutant. These results indicate that an efflux-based mechanism plays a role in reduced tigecycline susceptibility in Acinetobacter.

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5-Arylideneimidazolones with Amine at Position 3 as Potential Antibiotic Adjuvants against Multidrug Resistant Bacteria

Molecules ◽

10.3390/molecules24030438 ◽

2019 ◽

Vol 24 (3) ◽

pp. 438 ◽

Cited By ~ 3

Author(s):

Aneta Kaczor ◽

Karolina Witek ◽

Sabina Podlewska ◽

Joanna Czekajewska ◽

Annamaria Lubelska ◽

...

Keyword(s):

Efflux Pump ◽

Enterobacter Aerogenes ◽

Multidrug Resistant ◽

Resistant Bacteria ◽

Aromatic Rings ◽

Efflux Pump Inhibitor ◽

Fold Reduction ◽

Dual Action ◽

New Compounds

Searching for new chemosensitizers of bacterial multidrug resistance (MDR), chemical modifications of (Z)-5-(4-chlorobenzylidene)-2-(4-methylpiperazin-1-yl)-3H-imidazol-4(5H)-one (6) were performed. New compounds (7–17), with fused aromatic rings at position 5, were designed and synthesized. Crystallographic X-ray analysis proved that the final compounds (7–17) were substituted with tertiary amine-propyl moiety at position 3 and primary amine group at 2 due to intramolecular Dimroth rearrangement. New compounds were evaluated on their antibiotic adjuvant properties in either Gram-positive or Gram-negative bacteria. Efflux pump inhibitor (EPI) properties towards the AcrAB-TolC pump in Enterobacter aerogenes (EA289) were investigated in the real-time efflux (RTE) assay. Docking and molecular dynamics were applied to estimate an interaction of compounds 6–17 with penicillin binding protein (PBP2a). In vitro ADME-Tox properties were evaluated for compound 9. Most of the tested compounds reduced significantly (4-32-fold) oxacillin MIC in highly resistant MRSA HEMSA 5 strain. The anthracene-morpholine derivative (16) was the most potent (32-fold reduction). The tested compounds displayed significant EPI properties during RTE assay (37–97%). The naphthyl-methylpiperazine derivative 9 showed the most potent “dual action” of both oxacillin adjuvant (MRSA) and EPI (E. aerogenes). Molecular modeling results suggested the allosteric mechanism of action of the imidazolones, which improved binding of oxacillin in the PBP2a active site in MRSA.

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MgrB Inactivation Is Responsible for Acquired Resistance to Colistin in Enterobacter hormaechei subsp. steigerwaltii

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00128-20 ◽

2020 ◽

Vol 64 (6) ◽

Cited By ~ 2

Author(s):

Amel Mhaya ◽

Dominique Bégu ◽

Slim Tounsi ◽

Corinne Arpin

Keyword(s):

Acquired Resistance ◽

Regulatory System ◽

Multidrug Resistant ◽

Negative Regulator ◽

Clinical Strain ◽

Sequencing Analysis ◽

Content Type ◽

Health Care Associated Infections ◽

Enterobacter Hormaechei

ABSTRACT Multidrug-resistant strains belonging to the Enterobacter cloacae complex (ECC) group, and especially those belonging to clusters C-III, C-IV, and C-VIII, have increasingly emerged as a leading cause of health care-associated infections, with colistin used as one of the last lines of treatment. However, colistin-resistant ECC strains have emerged. The aim of this study was to prove that MgrB, the negative regulator of the PhoP/PhoQ two-component regulatory system, is involved in colistin resistance in ECC of cluster C-VIII, formerly referred to as Enterobacter hormaechei subsp. steigerwaltii. An in vitro mutant (Eh22-Mut) was selected from a clinical isolate of Eh22. The sequencing analysis of its mgrB gene showed the presence of one nucleotide deletion leading to the formation of a truncated protein of six instead of 47 amino acids. The wild-type mgrB gene from Eh22 and that of a clinical strain of Klebsiella pneumoniae used as controls were cloned, and the corresponding recombinant plasmids were used for complementation assays. The results showed a fully restored susceptibility to colistin and confirmed for the first time that mgrB gene expression plays a key role in acquired resistance to colistin in ECC strains.

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The Efflux Pump Inhibitor Timcodar Improves the Potency of Antimycobacterial Agents

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04271-14 ◽

2014 ◽

Vol 59 (3) ◽

pp. 1534-1541 ◽

Cited By ~ 24

Author(s):

Trudy H. Grossman ◽

Carolyn M. Shoen ◽

Steven M. Jones ◽

Peter L. Jones ◽

Michael H. Cynamon ◽

...

Keyword(s):

Mouse Model ◽

Efflux Pump ◽

Fold Increase ◽

Broth Culture ◽

Efflux Pump Inhibitor ◽

Content Type ◽

Combination Studies ◽

Drug Dependent

ABSTRACTPrevious studies indicated that inhibition of efflux pumps augments tuberculosis therapy. In this study, we used timcodar (formerly VX-853) to determine if this efflux pump inhibitor could increase the potency of antituberculosis (anti-TB) drugs againstMycobacterium tuberculosisinin vitroandin vivocombination studies. When used alone, timcodar weakly inhibitedM. tuberculosisgrowth in broth culture (MIC, 19 μg/ml); however, it demonstrated synergism in drug combination studies with rifampin, bedaquiline, and clofazimine but not with other anti-TB agents. WhenM. tuberculosiswas cultured in host macrophage cells, timcodar had about a 10-fold increase (50% inhibitory concentration, 1.9 μg/ml) in the growth inhibition ofM. tuberculosisand demonstrated synergy with rifampin, moxifloxacin, and bedaquiline. In a mouse model of tuberculosis lung infection, timcodar potentiated the efficacies of rifampin and isoniazid, conferring 1.0 and 0.4 log10reductions in bacterial burden in lung, respectively, compared to the efficacy of each drug alone. Furthermore, timcodar reduced the likelihood of a relapse infection when evaluated in a mouse model of long-term, chronic infection with treatment with a combination of rifampin, isoniazid, and timcodar. Although timcodar had no effect on the pharmacokinetics of rifampin in plasma and lung, it did increase the plasma exposure of bedaquiline. These data suggest that the antimycobacterial drug-potentiating activity of timcodar is complex and drug dependent and involves both bacterial and host-targeted mechanisms. Further study of the improvement of the potency of antimycobacterial drugs and drug candidates when used in combination with timcodar is warranted.

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The Impact of an Efflux Pump Inhibitor on the Activity of Free and Liposomal Antibiotics against Pseudomonas aeruginosa

Pharmaceutics ◽

10.3390/pharmaceutics13040577 ◽

2021 ◽

Vol 13 (4) ◽

pp. 577

Author(s):

Douweh Leyla Gbian ◽

Abdelwahab Omri

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Efflux Pump ◽

Antimicrobial Activities ◽

Dilution Method ◽

Efflux Pump Inhibitor ◽

Signal Production ◽

Lung Infections ◽

The Impact ◽

Microbroth Dilution

The eradication of Pseudomonas aeruginosa in cystic fibrosis patients has become continuously difficult due to its increased resistance to treatments. This study assessed the efficacy of free and liposomal gentamicin and erythromycin, combined with Phenylalanine arginine beta-naphthylamide (PABN), a broad-spectrum efflux pump inhibitor, against P. aeruginosa isolates. Liposomes were prepared and characterized for their sizes and encapsulation efficiencies. The antimicrobial activities of formulations were determined by the microbroth dilution method. Their activity on P. aeruginosa biofilms was assessed, and the effect of sub-inhibitory concentrations on bacterial virulence factors, quorum sensing (QS) signals and bacterial motility was also evaluated. The average diameters of liposomes were 562.67 ± 33.74 nm for gentamicin and 3086.35 ± 553.95 nm for erythromycin, with encapsulation efficiencies of 13.89 ± 1.54% and 51.58 ± 2.84%, respectively. Liposomes and PABN combinations potentiated antibiotics by reducing minimum inhibitory and bactericidal concentrations by 4–32 fold overall. The formulations significantly inhibited biofilm formation and differentially attenuated virulence factor production as well as motility. Unexpectedly, QS signal production was not affected by treatments. Taken together, the results indicate that PABN shows potential as an adjuvant of liposomal macrolides and aminoglycosides in the management of lung infections in cystic fibrosis patients.

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Staphylococcus aureus Mutants Isolated via Exposure to Nonfluorinated Quinolones: Detection of Known and Unique Mutations

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.12.3422-3426.2001 ◽

2001 ◽

Vol 45 (12) ◽

pp. 3422-3426 ◽

Cited By ~ 10

Author(s):

Siddhartha Roychoudhury ◽

Tracy L. Twinem ◽

Kelly M. Makin ◽

Mark A. Nienaber ◽

Chuiying Li ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Sequence Analysis ◽

Efflux Pump ◽

Serial Passage ◽

Efflux Pump Inhibitor ◽

In Vitro Development ◽

Development Of Resistance ◽

High Level ◽

Vitro Development

ABSTRACT The in vitro development of resistance to the new nonfluorinated quinolones (NFQs; PGE 9262932, PGE 4175997, and PGE 9509924) was investigated in Staphylococcus aureus. At concentrations two times the MIC, step 1 mutants were isolated more frequently with ciprofloxacin and trovafloxacin (9.1 × 10−8 and 5.7 × 10−9, respectively) than with the NFQs, gatifloxacin, or clinafloxacin (<5.7 × 10−10). Step 2 and step 3 mutants were selected via exposure of a step 1 mutant (selected with trovafloxacin) to four times the MICs of trovafloxacin and PGE 9262932. The step 1 mutant contained the known Ser80-Phe mutation in GrlA, and the step 2 and step 3 mutants contained the known Ser80-Phe and Ser84-Leu mutations in GrlA and GyrA, respectively. Compared to ciprofloxacin, the NFQs were 8-fold more potent against the parent and 16- to 128-fold more potent against the step 3 mutants. Mutants with high-level NFQ resistance (MIC, 32 μg/ml) were isolated by the spiral plater-based serial passage technique. DNA sequence analysis of three such mutants revealed the following mutations: (i) Ser84-Leu in GyrA and Glu84-Lys and His103-Tyr in GrlA; (ii) Ser-84Leu in GyrA, Ser52-Arg in GrlA, and Glu472-Val in GrlB; and (iii) Ser84-Leu in GyrA, Glu477-Val in GyrB, and Glu84-Lys and His103-Tyr in GrlA. Addition of the efflux pump inhibitor reserpine (10 μg/ml) resulted in 4- to 16-fold increases in the potencies of the NFQs against these mutants, whereas it resulted in 2-fold increases in the potencies of the NFQs against the parent.

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EXTH-31. BRAIN DISTRIBUTION, CLEARANCE AND TOXICITY EVALUATION OF SMALL-MOLECULE INHIBITORS FOLLOWING CONVECTION-ENHANCED DELIVERY TO THE BRAINSTEM

Neuro-Oncology ◽

10.1093/neuonc/noab196.670 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi170-vi170

Author(s):

Erica Power ◽

Juhee Oh ◽

Jonghoon Choi ◽

William Elmquist ◽

David Daniels

Keyword(s):

Small Molecule ◽

Drug Concentration ◽

Small Molecule Inhibitors ◽

Efflux Pump ◽

Sprague Dawley ◽

Convection Enhanced Delivery ◽

Efflux Pump Inhibitor ◽

Delivery Technique ◽

The Brain

Abstract BACKGROUND Diffuse midline gliomas (DMGs) harboring the H3K27M mutation are highly aggressive, fatal brainstem tumors that primarily occur in children. The blood-brain barrier (BBB) prevents numerous drugs from reaching CNS tumors, like DMG, at cytotoxic concentrations. Convection-enhanced delivery (CED) has emerged as a drug delivery technique that bypasses the BBB through a direct interstitial infusion under a pressure gradient. However, drug distribution and clearance from the brain following CED is poorly understood and has been cited as a potential reason for the lack of efficacy observed in prior clinical trials. OBJECTIVE The objective of this study was to understand how two small molecule inhibitors (alisertib, ponatinib) that inhibit cell growth and proliferation in DMG cells in vitro distribute and clear from the brain following CED to the brainstem. METHODS Sprague-dawley rats underwent a single 60mL CED infusion of drug to the brainstem (200mM alisertib, 10mM ponatinib) and were sacrificed 0.083, 1, 2, 4, 8 and 24 hours following the completion of the infusion. Brains were dissected and drug concentration was determined via HPLC analysis. RESULTS No rats showed any clinical or neurological signs of toxicity post-infusion. Both drugs showed significant differences in drug concentration based on anatomical brain region where higher concentrations were observed in the pons and cerebellum compared to the cortex. Drug half-life in the brain was ~0.5 hours for alisertib and ~1 hour for ponatinib, but this was not significantly increased following co-administration of elacridar, a BBB efflux pump inhibitor. CONCLUSIONS These results suggest that elimination of drugs from the brain in a complex, multifactorial mechanism that warrants further preclinical investigation prior to the initiation of a clinical trial.

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PUNICA GRANATUM RIND EXTRACT: ANTIBIOTIC POTENTIATOR AND EFFLUX PUMP INHIBITOR OF MULTIDRUG RESISTANT KLEBSIELLA PNEUMONIAE CLINICAL ISOLATES

Asian Journal of Pharmaceutical and Clinical Research ◽

10.22159/ajpcr.2017.v10i3.16000 ◽

2017 ◽

Vol 10 (3) ◽

pp. 191 ◽

Cited By ~ 2

Author(s):

Zumaana Rafiq ◽

Sreevidya Narasimhan ◽

Magesh Haridoss ◽

Rosy Vennila ◽

Rama Vaidyanathan

Keyword(s):

Klebsiella Pneumoniae ◽

Efflux Pump ◽

Ethanol Extract ◽

Punica Granatum ◽

Multidrug Resistant ◽

Synergistic Activity ◽

Sequential Fractionation ◽

Efflux Pump Inhibitor ◽

Fold Reduction ◽

Significant Area

ABSTRACTObjective: With a rise in multidrug resistant (MDR) bacterial isolates, search for antibiotics or compounds that could act synergistically with themis a significant area of research. Efflux-mediated resistance, in particular, is a great hurdle that needs to be overcome. In an effort to identify suchsynergistic compounds and potential efflux pump inhibitors (EPI), we analyzed the rind of Punica granatum (pomegranate) against MDR clinicalKlebsiella pneumoniae isolates.Methods: Sequential fractionation of P. granatum rind ethanol (PGR) extract was carried out to obtain hexane, butanol and water fractions.Antibacterial activity of the plant extracts was confirmed, and synergistic interaction with antibiotics was determined by the checkerboard assay. Gaschromatography-mass spectrometry (GC-MS) analysis was performed to identify the phytochemical constituents of the hexane extract. To study EPIactivity of the extracts, norfloxacin accumulation assay was carried out.Results: PGR ethanol extract was found to have synergistic activity with ciprofloxacin, levofloxacin, ceftazidime, cefoxitin, meropenem, and gentamicinresulting in fold decrease of minimum inhibitory concentration (MIC) ranging from 2 to 32 fold. The hexane fraction was found to have maximumsynergistic activity resulting in a 32-fold reduction of ciprofloxacin MIC followed by butanol and water fractions. The PGR ethanol extract was alsofound to have efflux inhibition activity by the norfloxacin accumulation assay. Of the sequential fractions, the butanol fraction had maximum effluxinhibition activity.Conclusion: Therefore, our study shows that PGR extract can potentiate the effect of antibiotics on MDR bacteria, and the mode of action is likely tobe due to EPI.Keywords: Punica granatum rind, Pomegranate, Synergy with antibiotics, Multidrug resistant, Klebsiella pneumoniae, Efflux pump inhibition.

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In Vitro Activity of the Novel Pleuromutilin Lefamulin (BC-3781) and Effect of Efflux Pump Inactivation on Multidrug-Resistant and Extensively Drug-Resistant Neisseria gonorrhoeae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01497-17 ◽

2017 ◽

Vol 61 (11) ◽

Cited By ~ 22

Author(s):

Susanne Jacobsson ◽

Susanne Paukner ◽

Daniel Golparian ◽

Jörgen S. Jensen ◽

Magnus Unemo

Keyword(s):

Efflux Pump ◽

Multidrug Resistant ◽

Cross Resistance ◽

Drug Resistant ◽

The Novel ◽

Extensively Drug Resistant ◽

Optimal Dosing ◽

And Performance ◽

Reference Strains

ABSTRACT We evaluated the activity of the novel semisynthetic pleuromutilin lefamulin, inhibiting protein synthesis and growth, and the effect of efflux pump inactivation on clinical gonococcal isolates and reference strains (n = 251), including numerous multidrug-resistant and extensively drug-resistant isolates. Lefamulin showed potent activity against all gonococcal isolates, and no significant cross-resistance to other antimicrobials was identified. Further studies of lefamulin are warranted, including in vitro selection and mechanisms of resistance, pharmacokinetics/pharmacodynamics, optimal dosing, and performance in randomized controlled trials.

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Curcumin as Efflux Pump Inhibitor Agent for Enhancement Treatment Against Multidrug Resistant Pseudomonas aeruginosa Isolates

Iraqi Journal of Science ◽

10.24996/ijs.2020.61.1.6 ◽

2020 ◽

pp. 59-67

Author(s):

Sulaiman D. Sulaiman ◽

Ghusoon A. Abdulhasan

Keyword(s):

Pseudomonas Aeruginosa ◽

Inhibitory Concentration ◽

Efflux Pump ◽

Multidrug Resistant ◽

Diffusion Method ◽

Disc Diffusion Method ◽

Efflux Pump Inhibitor ◽

Before And After ◽

Chromogenic Agar ◽

Susceptibility Patterns

Pseudomonas aeruginosa is considered as a developing opportunistic nosocomial pathogen and is well-known for its multidrug resistance that can be efficiently treated by a combination of antibiotics andefflux pump inhibitors (EPI). Therefore, the purpose of this study was to investigate the effect of curcumin as an EPI for the enhancement of the effectiveness of antibiotics against multidrug resistant (MDR) isolates ofP. aeruginosa. Susceptibility patterns of suspected bacteria was determined using the disc diffusion method andresistant bacteria were identified using chromogenic agar and 16S rDNA. The effectsof curcuminon the enhancement of antibiotics’s activity was evaluated usingthe broth microdilution method.The susceptibility patterns for 50 (67.6%) suspectedP. aeruginosaisolates showed that 36 (72%) of these isolateswere resistant to one of the used antibiotics,whereasonly 21 (42%) were MDR. The highest percentage of resistance was observedtoceftazidime (66%) followed by ciprofloxacin and levofloxacin (40%). Only 35 isolates were specified by chromogenic agar and 16S rDNAas P. aeruginosa.The minimal inhibitory concentration (MIC) of 35 isolates for ciprofloxacin resistant was between 4 and128 µg/ml while for ceftazidime was between 64and 512 µg/ml. After the addition of 50 μg/ml curcumin with ciprofloxacin, there wasa significant increase in the sensitivity (p≤ 0.01) of 13 MDR P.aeroginosa isolates whereas no differences in the sensitivity to ceftazidime were recorded before and after addition ofcurcumin. In conclusion, the results of this study show that curcumin can decrease the MIC value of ciprofloxacin in MDR isolates of P. aeruginosaand can be used as a native compound to enhance the treatment of resistant isolates with ciprofloxacin.

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Association of Efflux Pump and OMPs with Antibiotic Susceptibility Among ESBL-Producing Klebsiella Pneumoniae Clinical Strains in Malaysia

10.21203/rs.3.rs-375267/v1 ◽

2021 ◽

Author(s):

Golnaz Mobasseri ◽

Thong Kwai Lin ◽

Cindy Shuan Ju Teh

Keyword(s):

Antibiotic Resistance ◽

Klebsiella Pneumoniae ◽

Antibiotic Susceptibility ◽

Efflux Pump ◽

Outer Membrane Proteins ◽

Multidrug Resistant ◽

Efflux Pump Inhibitor ◽

Pump System ◽

Extended Spectrum Beta Lactamases ◽

Amoxicillin Clavulanate

Abstract Multidrug-resistant (MDR) Klebsiella pneumoniae (K. pneumoniae) poses a serious public health threat. K. pneumoniae strains that produce extended-spectrum beta-lactamases (ESBL) are becoming increasingly reported in nosocomial and community-acquired infections. Besides resistance genes, integrons, and plasmids, altered membrane permeability caused by porin loss and energy-dependent efflux have also contributed to antibiotic resistance in K. pneumoniae. The objective of this study was to determine the correlation between the reduction of antibiotic susceptibility and overexpression of efflux pump as well as the lack of outer membrane proteins (OMPs) among clinical ESBLs resistant K. pneumoniae. The expression levels of ramA, acrA, ompK35 and ompK36 in 12 MDR K. pneumoniae strains with varying MICs levels were analyzed using quantitative real time-Polymerase Chain Reaction (qRT-PCR). The role of efflux pump on antibiotic resistance was also studied by using minimum inhibitory concentration (MICs) method with//without efflux pump inhibitor. The result indicated that the strains with highest resistance to cefotaxime showed the lowest level of ompK35 and ompK36 genes expression while the strains with lowest MIC level of resistance to cefotaxime showed the highest level of expression of acrA and ramA. Our finding also revealed the effect of efflux pump inhibitor phenyl-arginine-b-naphthylamide (PAβN) on the MIC levels of ceftazidime, amoxicillin-clavulanate and cefotaxime which were significantly reduced around 1–7 folds MIC levels. These results suggest that Efflux pump system and deficiently of OMPs contributing role in antibiotic susceptibility which should be taken seriously to prevent the treatment failure due to antimicrobial resistance.

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