scholarly journals Characterization of AreABC, an RND-type efflux system involved in antimicrobial resistance of Aliarcobacter butzleri

2021 ◽  
Author(s):  
Susana Ferreira ◽  
Ana L. Silva ◽  
Joana Tomás ◽  
Cristiana Mateus ◽  
Fernanda Domingues ◽  
...  
Keyword(s):  
Antimicrobial Agents ◽  
Efflux Pump ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Efflux System ◽  
Erythromycin Resistance ◽  
Efflux Pump Inhibitor ◽  
Underlying Mechanisms ◽  
Multiple Drugs ◽  
Increased Susceptibility

Aliarcobacter butzleri is an emergent enteropathogen for which resistance to several classes of antimicrobial agents has been described, although the underlying mechanisms have been poorly addressed. We aimed to evaluate the contribution of the resistance-nodulation-division-type (RND) efflux system, AreABC, to drug resistance in A. butzleri . A. butzleri strains were first tested against several antimicrobials, with and without an efflux pump inhibitor. Then, erythromycin resistant strains were screened for the presence of a premature stop codon in a putative transcriptional regulator of the AreABC system, areR . Lastly, antimicrobial susceptibility and ethidium bromide (EtBr) accumulation were evaluated using an areB -knockout strain and a strain overexpressing the AreABC system through areR truncation. The presence of the efflux pump inhibitor resulted in increased susceptibility to most of the antimicrobials tested. A correlation between erythromycin resistance and the presence of premature stop codons in areR was observed. The truncation of areR resulted in increased expression of the AreABC system and decreased susceptibility to various antimicrobials. In contrast, areB inactivation resulted in increased susceptibility and a higher intracellular accumulation of EtBr. In conclusion, the AreABC efflux pump plays a role in the resistance of A. butzleri to multiple drugs and is regulated by a putative transcriptional repressor areR . Our results support the importance of efflux pumps in this bacterium's resistance to major classes of antibiotics and other antimicrobials.

scholarly journals Detection of efflux pump CmeABC in enrofloxacin resistant Campylobacter spp. strains isolated from broiler chickens (Gallus gallus domesticus) in the state of Rio de Janeiro, Brazil

2019 ◽  
Vol 39 (9) ◽  
pp. 728-733
Author(s):  
Regina J. Nascimento ◽  
Beatriz S. Frasão ◽  
Thomas S. Dias ◽  
Elmiro R. Nascimento ◽  
Louise S.B. Tavares ◽  
...  
Keyword(s):  
Rio De Janeiro ◽  
Broiler Chickens ◽  
Bacterial Resistance ◽  
Antimicrobial Agents ◽  
Efflux Pump ◽  
Metropolitan Region ◽  
Efflux System ◽  
Efflux Pump Inhibitor ◽  
Resistant Strains ◽  
Campylobacter Spp

ABSTRACT: Fowls are the main reservoirs of the highly important food-originating pathogen called Campylobacter spp. and broilers’ meat and byproducts are the main vehicles of this microorganism. Increasing of Campylobacter spp. resistant strains to fluorquinolones, an antimicrobial class often employed in poultry farming and in human medicine has become a great concern to poultry breeders. In fact, several studies evaluated increasing bacterial resistance against these antimicrobial agents. The role of CmeABC efflux system has been underscored among the resistance mechanisms in Campylobacter spp. to fluorquinolones. This study investigated the occurrence of CmeABC efflux pump in 81 and 78 enrofloxacin resistant strains of Campylobacter jejuni and C. coli respectively, isolated from broilers collected from six abattoirs situated at São José do Vale do Rio Preto/RJ poultry center and from two commercial abattoirs situated at Metropolitan Region of Rio de Janeiro, from 2013 to 2016. The resistance to enrofloxacin was assessed by agar dilution to determine minimum inhibitory concentration (MIC). The CmeABC efflux system was investigated through the detection of genes genes cmeA, cmeB and cmeC by PCR. The activity of CmeABC efflux pump was investigated in 20 strains by using the efflux pump inhibitor Phenylalanine-Arginine β-Naphthylamide (PAβN). The three genes cmeA, cmeB and cmeC were detected in 94.3% of the strains (C. jejuni = 80 and C. coli = 70), whereas the system was absent or incomplete in 5.7% of strains (C. jejuni = 1 and C. coli = 8). MIC varied between 0.5μg/ml and 64μg/ml, and 88.7% of strains were enrofloxacin resistant and 11.3% featuring intermediate resistance. The inhibition of the efflux pump by PAβN reduced the MIC to enrofloxacin up to eight times in fifteen strains (75%). These results indicate that this system is frequent and active in Campylobacter spp. Resistant strains in the presence of enrofloxacin.

scholarly journals Efflux Pump-Mediated Intrinsic Drug Resistance in Mycobacterium smegmatis

2004 ◽  
Vol 48 (7) ◽  
pp. 2415-2423 ◽  
Author(s):  
Xian-Zhi Li ◽  
Li Zhang ◽  
Hiroshi Nikaido
Keyword(s):  
Drug Resistance ◽  
Ethidium Bromide ◽  
Mycobacterium Smegmatis ◽  
Efflux Pump ◽  
Upstream Region ◽  
Drug Efflux ◽  
Intact Cells ◽  
Intrinsic Drug Resistance ◽  
Multiple Drugs ◽  
Increased Susceptibility

ABSTRACT The Mycobacterium smegmatis genome contains many genes encoding putative drug efflux pumps. Yet with the exception of lfrA, it is not clear whether these genes contribute to the intrinsic drug resistance of this organism. We showed first by reverse transcription (RT)-PCR that several of these genes, including lfrA as well as the homologues of Mycobacterium tuberculosis Rv1145, Rv1146, Rv1877, Rv2846c (efpA), and Rv3065 (mmr and emrE), were expressed at detectable levels in the strain mc2155. Null mutants each carrying an in-frame deletion of these genes were then constructed in M. smegmatis. The deletions of the lfrA gene or mmr homologue rendered the mutant more susceptible to multiple drugs such as fluoroquinolones, ethidium bromide, and acriflavine (two- to eightfold decrease in MICs). The deletion of the efpA homologue also produced increased susceptibility to these agents but unexpectedly also resulted in decreased susceptibility to rifamycins, isoniazid, and chloramphenicol (two- to fourfold increase in MICs). Deletion of the Rv1877 homologue produced some increased susceptibility to ethidium bromide, acriflavine, and erythromycin. The upstream region of lfrA contained a gene encoding a putative TetR family transcriptional repressor, dubbed LfrR. The deletion of lfrR elevated the expression of lfrA and produced higher resistance to multiple drugs. Multidrug-resistant single-step mutants, independent of LfrA and attributed to a yet-unidentified drug efflux pump (here called LfrX), were selected in vitro and showed decreased accumulation of norfloxacin, ethidium bromide, and acriflavine in intact cells. Finally, use of isogenic β-lactamase-deficient strains showed the contribution of LfrA and LfrX to resistance to certain β-lactams in M. smegmatis.

scholarly journals Rescued chlorhexidine activity by resveratrol against carbapenem-resistant Acinetobacter baumannii via down-regulation of AdeB efflux pump

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0243082
Author(s):  
Uthaibhorn Singkham-in ◽  
Paul G. Higgins ◽  
Dhammika Leshan Wannigama ◽  
Parichart Hongsing ◽  
Tanittha Chatsuwan
Keyword(s):  
Acinetobacter Baumannii ◽  
Ethidium Bromide ◽  
Antimicrobial Agents ◽  
Efflux Pump ◽  
Intracellular Accumulation ◽  
Efflux Pump Inhibitor ◽  
Carbapenem Resistant ◽  
Efflux Pump Inhibition ◽  
Synergistic Mechanism ◽  
Time Kill

The aim of this study was to determine the activity and synergistic mechanisms of resveratrol in combination with chlorhexidine against carbapenem-resistant Acinetobacter baumannii clinical isolates. The activity of resveratrol plus antimicrobial agents was determined by checkerboard and time-kill assay against carbapenem-resistant A. baumannii isolated from patients at the King Chulalongkorn Memorial Hospital, Bangkok, Thailand. Overexpression of efflux pumps that mediates chlorhexidine susceptibility was characterized by the ethidium bromide accumulation assay. The effect of resveratrol on the expression of efflux pump genes (adeB, adeJ, adeG abeS, and aceI) and the two-component regulators, adeR and adeS was determined by RT-qPCR. The combination of resveratrol and chlorhexidine resulted in strong synergistic and bactericidal activity against carbapenem-resistant A. baumannii. Up-regulation of adeB and aceI was induced by chlorhexidine. However, the addition of resveratrol increased chlorhexidine susceptibility with increased intracellular accumulation of ethidium bromide in A. baumannii indicating that resveratrol acts as an efflux pump inhibitor. Expression of adeB was significantly reduced in the combination of resveratrol with chlorhexidine indicating that resveratrol inhibits the AdeB efflux pump and restores chlorhexidine effect on A. baumannii. In conclusion, reduced adeB expression in A. baumannii was mediated by resveratrol suggesting that AdeB efflux pump inhibition contributes to the synergistic mechanism of resveratrol with chlorhexidine. Our finding highlights the potential importance of resveratrol in clinical applications.

scholarly journals CmeABC Functions as a Multidrug Efflux System in Campylobacter jejuni

2002 ◽  
Vol 46 (7) ◽  
pp. 2124-2131 ◽  
Author(s):  
Jun Lin ◽  
Linda Overbye Michel ◽  
Qijing Zhang
Keyword(s):  
Campylobacter Jejuni ◽  
Antimicrobial Agents ◽  
Efflux Pump ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Intrinsic Resistance ◽  
Multidrug Efflux Pump ◽  
Efflux Pump Inhibitor ◽  
Gram Negative ◽  
Multidrug Efflux Pumps

ABSTRACT Campylobacter jejuni, a gram-negative organism causing gastroenteritis in humans, is increasingly resistant to antibiotics. However, little is known about the drug efflux mechanisms in this pathogen. Here we characterized an efflux pump encoded by a three-gene operon (designated cmeABC) that contributes to multidrug resistance in C. jejuni 81-176. CmeABC shares significant sequence and structural homology with known tripartite multidrug efflux pumps in other gram-negative bacteria, and it consists of a periplasmic fusion protein (CmeA), an inner membrane efflux transporter belonging to the resistance-nodulation-cell division superfamily (CmeB), and an outer membrane protein (CmeC). Immunoblotting using CmeABC-specific antibodies demonstrated that cmeABC was expressed in wild-type 81-176; however, an isogenic mutant (9B6) with a transposon insertion in the cmeB gene showed impaired production of CmeB and CmeC. Compared to wild-type 81-176, 9B6 showed a 2- to 4,000-fold decrease in resistance to a range of antibiotics, heavy metals, bile salts, and other antimicrobial agents. Accumulation assays demonstrated that significantly more ethidium bromide and ciprofloxacin accumulated in mutant 9B6 than in wild-type 81-176. Addition of carbonyl cyanide m-chlorophenylhydrazone, an efflux pump inhibitor, increased the accumulation of ciprofloxacin in wild-type 81-176 to the level of mutant 9B6. PCR and immunoblotting analysis also showed that cmeABC was broadly distributed in various C. jejuni isolates and constitutively expressed in wild-type strains. Together, these findings formally establish that CmeABC functions as a tripartite multidrug efflux pump that contributes to the intrinsic resistance of C. jejuni to a broad range of structurally unrelated antimicrobial agents.

scholarly journals Yersinia pestisacrAB-tolCin Antibiotic Resistance and Virulence

2011 ◽  
Vol 56 (2) ◽  
pp. 1120-1123 ◽  
Author(s):  
Ida M. Lister ◽  
Connor Raftery ◽  
Joan Mecsas ◽  
Stuart B. Levy
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Yersinia Pestis ◽  
Pathogenic Bacteria ◽  
Efflux Pump ◽  
Efflux Pump Inhibitor ◽  
Content Type ◽  
Increased Susceptibility

ABSTRACTThe efflux pump AcrAB is important in the antibiotic resistance and virulence of several pathogenic bacteria. We report that deletion of theYersinia pestisAcrAB-TolC homolog leads to increased susceptibility to diverse substrates, including, though unlike inEscherichia coli, the aminoglycosides. Neither is theY. pestispump affected by the efflux pump inhibitor phenylalanine-arginine beta-naphthylamide. In mouse plague models, pump deletion does not have a significant effect on tissue colonization.

scholarly journals High Frequency of AdeA, AdeB and AdeC Genes among Acinetobacter baumannii Isolates

2020 ◽  
Author(s):  
Reza RANJBAR ◽  
Shahin ZAYERI ◽  
Davoud AFSHAR
Keyword(s):  
Acinetobacter Baumannii ◽  
Antimicrobial Agents ◽  
Susceptibility Testing ◽  
Efflux Pump ◽  
Multidrug Transporter ◽  
Efflux System ◽  
Pcr Assay ◽  
Multiplex Pcr Assay ◽  
High Prevalence ◽  
Imipenem Resistance

Background: Efflux pumps are involved in resistance of Acinetobacter baumannii isolates to antimicrobial agents. AdeABC efflux pump is one of the RND superfamily efflux pump and consists of adeA (membrane fusion), adeB (multidrug transporter) and adeC (outer membrane) genes. In this study, the frequency of adeA, adeB and adeC genes among A. baumannii isolates with resistance to erythromycin, trimethoprim, meropenem and imipenem was investigated. Methods: Overall, 79 strains of A. baumannii were isolated from patients admitted to two major hospitals in Tehran during 2016. Antibiotic susceptibility testing was determined by disc diffusion and microdilution methods according to Clinical and Laboratory Standards Institute (CLSI) guideline. The presence of adeA, adeB and adeC genes was also determined using Multiplex PCR assay. Results: The highest and the lowest resistance among A. baumannii isolates were to trimethoprim (93%) and erythromycin (53%), respectively. The frequency of adeA, adeB and adeC genes was 96.2%, 96.2% and 91.1 % respectively. There was a significant relationship between imipenem resistance and presence of efflux pump genes (P<0.05). Conclusion: According to the high prevalence of the AdeABC efflux system genes, it may involve in resistance of clinical isolates of A. baumannii to imipenem, especially.  

scholarly journals Ciprofloxacin-Resistant Salmonella enterica Serovar Typhimurium Strains Are Difficult To Select in the Absence of AcrB and TolC

2006 ◽  
Vol 50 (1) ◽  
pp. 38-42 ◽  
Author(s):  
Vito Ricci ◽  
Peter Tzakas ◽  
Anthony Buckley ◽  
Nick C. Coldham ◽  
Laura J. V. Piddock
Keyword(s):  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Efflux Pump ◽  
Multiple Antibiotic Resistance ◽  
Efflux System ◽  
Efflux Pump Inhibitor ◽  
Serovar Typhimurium ◽  
Resistant Mutants ◽  
Selection Of ◽  
Selection Of Resistance

ABSTRACT It has been proposed that lack of a functional efflux system(s) will lead to a lower frequency of selection of resistance to fluoroquinolones and other antibiotics. We constructed five strains of Salmonella enterica serovar Typhimurium SL1344 that lacked efflux gene components of resistance nodulation cell division pumps (acrB, acrD, acrF, acrBacrF, and tolC) plus three strains that lack genes that effect efflux gene expression (marA, soxS, and ramA) and a hypermutable strain (mutS::aph). Strains were exposed to ciprofloxacin at 2× the MIC in agar, in the presence and absence of Phe-Arg-β-naphthylamide, an efflux pump inhibitor. Mutants were selected from all strains except those lacking acrB, tolC, or acrBacrF. For strains from which mutants were selected, there were no significant differences between the frequencies of resistance. Except for mutants of the ramA::aph strain, two phenotypes arose: resistance to quinolones only and multiple antibiotic resistance (MAR). ramA::aph mutants were resistant to quinolones only, suggesting a role for ramA in MAR in S. enterica serovar Typhimurium. Phe-Arg-β-naphthylamide (20 μg/ml) had no effect on the frequencies of resistance or ciprofloxacin MICs. In conclusion, functional AcrB and TolC in S. enterica serovar Typhimurium are important for the selection of ciprofloxacin-resistant mutants.

scholarly journals Limited MDR efflux pump overexpression among multidrug-resistant Escherichia coli of ST131

2021 ◽  
Author(s):  
Johannes Camp ◽  
Sabine Schuster ◽  
Martina Vavra ◽  
Tobias Schweigger ◽  
John W. A. Rossen ◽  
...  
Keyword(s):  
Antimicrobial Agents ◽  
Efflux Pump ◽  
Principal Component ◽  
Efflux Pumps ◽  
Multidrug Resistant ◽  
Gram Negative Bacteria ◽  
E Coli ◽  
Enhanced Expression ◽  
And Control ◽  
Increased Susceptibility

Gram-negative bacteria partly rely on efflux pumps to facilitate growth under stressful conditions and to increase resistance to a wide variety of commonly used drugs. In recent years E. coli ST131 has emerged as a major cause of extraintestinal infection frequently exhibiting an MDR phenotype. The contribution of efflux to MDR in emerging E. coli MDR clones however, is not well studied. We characterized strains from an international collection of clinical MDR-E. coli isolates by MIC testing with and without the addition of the AcrAB-TolC efflux inhibitor 1-(1-naphthylmethyl)-piperazine (NMP). MIC data for 6 antimicrobial agents and their reversion by NMP were analyzed by Principal Component Analysis (PCA). PCA revealed a group of 17/34 MDR-E. coli exhibiting increased susceptibility to treatment with NMP suggesting an enhanced contribution of efflux pumps to antimicrobial resistance in these strains (termed “enhanced efflux phenotype” [EEP]). Only 1/17 EEP strains versus 12/17 non-EEP MDR strains belonged to the ST131 clonal group. Whole-genome sequencing revealed marked differences in efflux-related genes between EEP and control strains, with the majority of notable amino-acid substitutions occurring in AcrR, MarR and SoxR. qRT-PCR of multiple efflux-related genes showed significant overexpression of the AcrAB-TolC-system in EEP strains, whereas in the remaining strains we found enhanced expression of alternative efflux proteins. We conclude that a proportion of MDR E. coli exhibit an EEP, which is linked to an overexpression of the AcrAB-TolC-efflux-pump and a distinct array of genomic variations. Members of ST131, although highly successful, are less likely to exhibit the EEP.

scholarly journals Perturbed structural dynamics underlie inhibition and altered efflux of the multidrug resistance pump AcrB

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Eamonn Reading ◽  
Zainab Ahdash ◽  
Chiara Fais ◽  
Vito Ricci ◽  
Xuan Wang-Kan ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Structural Dynamics ◽  
Antimicrobial Agents ◽  
Efflux Pump ◽  
Binding Pocket ◽  
Drug Binding ◽  
Multidrug Efflux Pump ◽  
Transport Pathway ◽  
Efflux Pump Inhibitor ◽  
Wide Range

Abstract Resistance–nodulation–division efflux pumps play a key role in inherent and evolved multidrug resistance in bacteria. AcrB, a prototypical member of this protein family, extrudes a wide range of antimicrobial agents out of bacteria. Although high-resolution structures exist for AcrB, its conformational fluctuations and their putative role in function are largely unknown. Here, we determine these structural dynamics in the presence of substrates using hydrogen/deuterium exchange mass spectrometry, complemented by molecular dynamics simulations, and bacterial susceptibility studies. We show that an efflux pump inhibitor potentiates antibiotic activity by restraining drug-binding pocket dynamics, rather than preventing antibiotic binding. We also reveal that a drug-binding pocket substitution discovered within a multidrug resistant clinical isolate modifies the plasticity of the transport pathway, which could explain its altered substrate efflux. Our results provide insight into the molecular mechanism of drug export and inhibition of a major multidrug efflux pump and the directive role of its dynamics.

scholarly journals Cloning, Nucleotide Sequencing, and Analysis of the AcrAB-TolC Efflux Pump of Enterobacter cloacae and Determination of Its Involvement in Antibiotic Resistance in a Clinical Isolate

2007 ◽  
Vol 51 (9) ◽  
pp. 3247-3253 ◽  
Author(s):  
Astrid Pérez ◽  
Delia Canle ◽  
Cristina Latasa ◽  
Margarita Poza ◽  
Alejandro Beceiro ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Clinical Isolate ◽  
Antimicrobial Agents ◽  
Efflux Pump ◽  
Enterobacter Aerogenes ◽  
Enterobacter Cloacae ◽  
Pcr Analysis ◽  
Nucleotide Sequencing ◽  
Efflux Pump Inhibitor ◽  
Antibiotic Resistance Profile

ABSTRACT Enterobacter cloacae is an emerging clinical pathogen that may be responsible for nosocomial infections. Management of these infections is often difficult, owing to the high frequency of strains that are resistant to disinfectants and antimicrobial agents in the clinical setting. Multidrug efflux pumps, especially those belonging to the resistance-nodulation-division family, play a major role as a mechanism of antimicrobial resistance in gram-negative pathogens. In the present study, we cloned and sequenced the genes encoding an AcrAcB-TolC-like efflux pump from an E. cloacae clinical isolate (isolate EcDC64) showing a broad antibiotic resistance profile. Sequence analysis showed that the acrR, acrA, acrB, and tolC genes encode proteins that display 79.8%, 84%, 88%, and 82% amino acid identities with the respective homologues of Enterobacter aerogenes and are arranged in a similar pattern. Deletion of the acrA gene to yield an AcrA-deficient EcDC64 mutant (EcΔacrA) showed the involvement of AcrAB-TolC in multidrug resistance in E. cloacae. However, experiments with an efflux pump inhibitor suggested that additional efflux systems also play a role in antibiotic resistance. Investigation of several unrelated isolates of E. cloacae by PCR analysis revealed that the AcrAB system is apparently ubiquitous in this species.

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